Patent Publication Number: US-2020284811-A1

Title: Bicarbonate biosensors, calcium biosensors, and uses thereof

Description:
RELATED APPLICATIONS 
     This application claims benefit of priority to U.S. Provisional Application No. 62/257,792, filed Nov. 20, 2015, U.S. Provisional Application No. 62/257,817, filed Nov. 20, 2015, and U.S. Provisional Application No. 62/257,796, filed Nov. 20, 2015, the entire contents of each of which are incorporated herein by reference. 
    
    
     INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING 
     The contents of the text file named “35327-526001WO_Sequence_Listing.txt”, which was created on Nov. 19, 2016 and is 466 KB in size, is hereby incorporated by reference in its entirety. 
     FIELD OF THE INVENTION 
     The present invention relates to compositions and methods for detecting and determining the concentrations of bicarbonate and calcium. 
     BACKGROUND 
     Bicarbonate levels are currently measured using either sample acidification in combination with a CO 2 -selective electrode, or alkalinization followed by a colorimetric coupled enzyme assay (Burtis 2012). Reagents and the modification of test samples is required in such methods. 
     Many fluorescent indicators have been developed to measure calcium levels within cells (Valeur 2012). However, these indicators are tuned for the micromolar range and therefore are not appropriate for measuring extracellular levels. 
     Improved sensors for bicarbonate and calcium are needed. 
     SUMMARY OF THE INVENTION 
     Aspects of the present subject matter provide improved biosensors that rapidly, reliably, and accurately detect and quantify ligands such as bicarbonate and calcium (i.e., Ca 2+ ) with significant advantages over previous systems. 
     The present disclosure also provides a biosensor for bicarbonate, comprising a reporter group that is attached to a bicarbonate-binding protein. The bicarbonate-binding protein includes a domain or region(s) that binds the bicarbonate. The domain or region involved in ligand binding is comprised of a plurality of residues, e.g., non-contiguous amino acids of the ligand-binding protein, which are contact points or sites of contact between the ligand and its cognate ligand-binding protein. The binding of bicarbonate to the bicarbonate-binding domain of the bicarbonate-binding protein causes a change in signaling by the reporter group. In various implementations, the biosensor may produce a signal when a bicarbonate is bound to the bicarbonate-binding domain that is not produced (and/or that is different from a signal that is produced) when the bicarbonate is absent from the bicarbonate-binding domain. 
     Also included are biosensors for calcium (i.e. Ca 2+ ) comprising a calcium-binding protein and an attached reporter group. Upon Ca 2+  binding to a calcium-binding domain of the calcium-binding protein, signaling of the reporter group changes. In various embodiments, the calcium-binding protein comprises a variant or mutant of a naturally occurring bicarbonate-binding protein. In certain embodiments, the calcium-binding protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions compared to a naturally occurring bicarbonate-binding protein. In some embodiments, the calcium-binding protein is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a bicarbonate-binding protein. In various embodiments, the calcium-binding protein has a Kd for bicarbonate that is at least about 5 mM, 10 mM, 25 mM, 50 mM, 75 mM, 100 mM, 500 mM, or 1000 mM higher than a corresponding naturally occurring bicarbonate-binding protein and/or Ca 2+ . 
     These biosensors have widespread utility including in clinical, industrial, food and beverage production and storage, and environmental settings. 
     A reporter group that transduces a detectable signal may be attached to the ligand-binding proteins (biosensors) described herein. The reporter group is attached to the ligand-binding protein so that a signal transduced by the reporter group when the ligand-binding protein is bound to ligand differs from a signal transduced by the reporter group when the ligand-binding protein is not bound to ligand. The proteins may be engineered to include a single cysteine to which the detectable label, e.g., a fluorophore is covalently attached. The biosensors are reagentless in that their monitoring mechanism requires neither additional substrates for a signal to develop, nor measurement of substrate consumption or product generation rates to determine ligand concentrations. 
     In some embodiments, the biosensor proteins include a second fluorophore, thereby permitting ratiometric sensing/detection of an analyte using establishing non-geometrically modulated Förster resonance energy transfer (ngmFRET). 
     Among the advantages of these fluorophore-containing protein constructs is their high durability. The constructs retain their ability to bind ligand, change shape and thus detect the analyte (such as bicarbonate or Ca 2+ ) (a) even when immobilized (directly or indirectly) onto a solid surface such as a bead, plate, or sheet; (b) even after desiccation (and subsequent reconstitution in a physiological buffer solution); (c) even when subjected to ambient conditions, e.g., conditions that can be encountered in storage and/or transportation; and (d) even when aged/stored for extended periods of time, e.g., weeks, months, or even years. Thus, the biosensors do not require refrigeration or a cold chain for distribution, permitting a wider range of applicability such as in-the-field use and reducing the cost of the sensor product. 
     For clinical applications, microliter volumes (e.g., less than 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or less than 10 μl) of a bodily fluid such as blood may be used. Moreover compared to conventional enzyme-based or antibody based assay systems, the results are achieved virtually instantaneously, e.g., 0.1-5 minutes, e.g., 0.1-1 minutes, or within 30-60 seconds. A further advantage is that the sensors consistently and reliably bind to and detect the analyte (bicarbonate or a Ca 2+ ) in complex fluids such as whole blood, plasma, serum, saliva, urine, and environmental fluids. Thus in a clinical setting, whole blood need not be processed, thereby reducing time and cost of the diagnostic procedure. Alternatively or in addition, the biosensors provided herein may be used to monitor ligand levels continuously. In a non-limiting example, one or more biosensors is immobilized at the tip of a thin optical fiber to construct a ligand-responsive optode. Such an optode can be introduced into the body (e.g., subcutaneously). The sensor may be in continuous contact with the sample, and excitation and emission light are passed to and from the immobilized sensor, respectively. Fluctuations in the ligand sample alter the dynamic equilibrium between the open and closed states of the ligand-binding protein, which is transduced into fluctuations of the fluorescent emission signal, by virtue of the sensing mechanism of the conjugated fluorophore. The emitted light intensities may be read by a reader connected to the optode. 
     In non-clinical situations, e.g., food and beverage composition (e.g., meat, canned food, dairy, nondairy, a fermented food, a fruit, a vegetable, a tuber, a starch, a grain, pasta, yogurt, soup, ice cream, a broth, a puree, a shake, a smoothie, a batter, a condiment, a sauce, a soft drink, a fountain beverage, water, coffee, tea, milk, a dairy-based beverages, soy-based beverage, an almond-based beverage, vegetable juice, fruit juice, a fruit juice-flavored drink, an energy drink, or an alcoholic beverage) production and/or storage, industrial, environmental (e.g., wetlands, rivers, streams, ponds, marine environments, wells, aquariums, pools, lakes, rivers, brooks, reservoirs, ground water, residential land, commercial/industrial land, agricultural land, or land abutting agricultural land), or commercial settings such as analysis of waste water, food or beverage production, or bioreactor/fermentation monitoring, the samples to be analyzed can be used directly upon sampling without further purification or processing, similarly reducing time and expense of the test. Moreover, the immobilized sensors need not be washed to remove unbound material following contacting the test sample with the sensors, because the unbound material (“contaminants”) do not materially affect the production of a precise, reliable detectable assay signal. 
     Included herein are bicarbonate and Ca 2+  biosensors that produce a dichromatic, ratiometric signal, i.e., the signal is defined as the quotient of the intensities at two independent wavelengths. The advantage of such a signal is that it provides an internally consistent reference. The self-calibrating nature of a ratiometric measurement removes the necessity for carrying out on-board calibration tests prior to each measurement. 
     Thus, reagentless, fluorescently responsive biosensors present a number of advantages over enzyme-based biosensors, including elimination of chemical transformations, elimination of substrate requirements, and self-calibration, which together lead to rapid response times, continuous monitoring capabilities, simple sample-handling, and lower cost due to simplified manufacturing and distribution processes. 
     Ligand-Binding Proteins 
     Aspects of the present subject matter provide biosensors comprising a ligand-binding protein that binds bicarbonate (i.e., a bicarbonate-binding protein). Variants of bicarbonate-binding proteins that have been selected, designed, or engineered to specifically bind Ca 2+  (i.e., calcium-binding proteins) are also included. The biosensors provided herein may have an affinity for ligand that falls within a range that is relevant for commercial, industrial, research, food and beverage, cell culture, environmental, and/or physiological (e.g., clinical or veterinarial) applications. 
     Typically, a natural bicarbonate-binding protein has a bicarbonate dissociation constant (K d ) of about 10 μM or less at room temperature. However, bicarbonate-binding proteins may be selected, designed, or engineered (e.g., via mutation) to have a different affinity for bicarbonate (e.g., to detect higher or lower levels of bicarbonate). In embodiments, a bicarbonate-binding protein has a K d  for bicarbonate in the micromolar, nanomolar, picomolar, or femtomolar range. In various embodiments, the bicarbonate-binding protein binds bicarbonate as a metal complex, e.g., as calcium (Ca II )-bicarbonate, or as iron (Fe III )-bicarbonate. In some embodiments, the bicarbonate-binding protein has a K d  for calcium (Ca II )-bicarbonate or iron (Fe III )-bicarbonate in the micromolar, nanomolar, picomolar, or femtomolar range. For example, a bicarbonate-binding protein may have a K d  for bicarbonate of at least about 0.00001 mM, 0.0001 mM, 0.001 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 55 mM, 6 mM 7M, 8 mM, 9M, 10 mM, II mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 75 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, or 1000 mM, and/or less than about 0.00001 mM, 0.0001 mM, 0.001 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 75 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, or 1000 mM. In various embodiments, the K d  for bicarbonate is as measured in a solution comprising 1 mM Ca 2+  or 10 μM Fe 3+ . In some embodiments, a bicarbonate-binding protein has a K d  for bicarbonate below, within, or above the normal range of bicarbonate in human blood. The normal blood bicarbonate concentration in humans is typically referred to as 22-29 mM but may vary by sex and age. Exemplary normal bicarbonate levels are as follows: 
     Males 
     12-24 months: 17-25 mM 
     3 years: 18-26 mM 
     4-5 years: 19-27 mM 
     6-7 years: 20-28 mM 
     8-17 years: 21-29 mM 
     &gt; or =18 years: 22-29 mM 
     Females 
     1-3 years: 18-25 mM 
     4-5 years: 19-26 mM 
     6-7 years: 20-27 mM 
     8-9 years: 21-28 mM 
     &gt; or =10 years: 22-29 mM 
     Aspects provide bicarbonate biosensors comprising bicarbonate-binding proteins with a K d  for bicarbonate within one of more of the ranges disclosed herein. 
     In various embodiments, a calcium-binding protein has a K for Ca 2+  in the millimolar, micromolar, nanomolar, picomolar, or femtomolar range. The normal physiological concentration of Ca 2+  in human blood is between 1.2-1.3 mM. The detection of much higher amounts/ranges is useful in, e.g., environmental, food and beverage, and industrial applications. For example, a calcium-binding protein may have a K d  for Ca 2+  of at least about 0.00001 mM, 0.0001 mM, 0.001 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, 2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM, 3 mM, 3.1 mM, 3.2 mM, 3.3 mM, 3.4 mM, 3.5 mM, 3.6 mM, 3.7 mM, 3.8 mM, 3.9 mM, 4 mM, 4.1 mM, 4.2 mM, 4.3 mM, 4.4 mM, 4.5 mM, 4.6 mM, 4.7 mM, 4.8 mM, 4.9 mM, 5 mM, 5.1 mM, 5.2 mM, 5.3 mM, 5.4 mM, 5.5 mM, 5.6 mM, 5.7 mM, 5.8 mM, 5.9 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, or 1000 mM, and/or less than about 0.00001 mM, 0.0001 mM, 0.001 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, 2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM, 3 mM, 3.1 mM, 3.2 mM, 3.3 mM, 3.4 mM, 3.5 mM, 3.6 mM, 3.7 mM, 3.8 mM, 3.9 mM, 4 mM, 4.1 mM, 4.2 mM, 4.3 mM, 4.4 mM, 4.5 mM, 4.6 mM, 4.7 mM, 4.8 mM, 4.9 mM, 5 mM, 5.1 mM, 5.2 mM, 5.3 mM, 5.4 mM, 5.5 mM, 5.6 mM, 5.7 mM, 5.8 mM, 5.9 mM, 6 mM, 7 mM 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM, 36 mM, 37 mM, 38 mM, 39 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, or 1000 mM. Aspects provide cation biosensors comprising calcium-binding proteins with a K d  for Ca 2+  within one of more of the ranges disclosed herein. 
     With respect to the present subject matter, K d  is the equilibrium dissociation constant between a ligand-binding protein and its ligand. K d  decreases with increasing affinity, and K d  may be used as an expression of affinity (the lower the value, the higher the affinity). The K d  value relates to the concentration of ligand required for detectable ligand-binding to occur and so the lower the K d  value (lower concentration required), the higher the affinity of the ligand-binding protein for the ligand. The K d  value corresponds to the ligand concentration at which the binding protein is 50% saturated. 
     
       
         
           
               
               
             
               
                   
               
               
                 K d  value 
                 Molar concentration 
               
               
                   
               
             
            
               
                 10 −1  to 10 −3   
                 Millimolar (mM) 
               
               
                 10 −4  to 10 −6   
                 Micromolar (μM) 
               
               
                 10 −7  to 10 −9   
                 Nanomolar (nM) 
               
               
                 10 −10  to 10 −12   
                 Picomolar (pM) 
               
               
                 10 −13  to 10 −15   
                 Femtomolar (fM) 
               
               
                   
               
            
           
         
       
     
     The ligand-binding proteins (as well as biosensors comprising the ligand-binding proteins) provided herein lack enzymatic activity and are not enzymes. As used herein, an “enzyme” is a protein that catalyzes a specific biochemical reaction. The ligand is not chemically altered (i.e., no chemical bond or atom of the ligand is added or removed) by the ligand-binding protein. Thus, when ligand dissociates from a ligand-binding protein described herein, the ligand contains the same chemical structure it had before it became bound to the ligand-binding protein. 
     The ligand-binding protein may comprise a naturally occurring protein or a protein that is modified compared to a naturally occurring protein. For example, the ligand-binding protein may comprise one or more mutations compared to a naturally occurring protein. In some embodiments, the naturally occurring protein is a naturally occurring counterpart of the ligand-binding protein (e.g., the ligand-binding protein is a mutant of the naturally occurring counterpart). 
     A “naturally occurring counterpart” of a mutant polypeptide is a polypeptide produced in nature from which the mutant polypeptide has been or may be derived (e.g., by one or more mutations). For example, the naturally occurring counterpart is an endogenous polypeptide produced by an organism in nature, wherein the endogenous polypeptide typically does not have one or more of the mutations present in the mutant polypeptide. For convenience and depending on context, a naturally occurring counterpart may be referred to herein for the purpose of comparison and to illustrate the location and/or presence of one or more mutations, binding activities, and/or structural features. 
     As used herein, a “mutation” is a difference between the amino acid sequence of a modified polypeptide/protein and a naturally occurring counterpart. A polypeptide having a mutation may be referred to as a “mutant.” Non-limiting examples of mutations include insertions, deletions, and substitutions. However, the term “mutation” excludes (i) the addition of amino acids to the N-terminus or C-terminus of a polypeptide, and (ii) the omission/deletion/replacement of a polypeptide&#39;s signal peptide (e.g., replacement with another signal peptide or with a methionine). 
     The addition of amino acids to the N-terminus or C-terminus of a protein via a peptide bond may be referred to herein as a “fusion” of the amino acids to the protein. Similarly, an exogenous protein fused to amino acids (e.g., another protein, a fragment, a tag, or a polypeptide moiety) at its N-terminus or C-terminus may be referred to as a “fusion protein.” The added amino acids may comprise a heterologous polypeptide, e.g., a polypeptide reporter group such as a fluorescent protein, a moiety that facilitates the isolation or modification of a polypeptide, or a moiety that facilitates the attachment of a polypeptide to a substrate or surface. As used herein, “heterologous” when referring to the added amino acids (e.g., a “polypeptide”) of a fusion protein indicates that the polypeptide is not naturally part of the protein to which it is fused in the fusion protein. For example, the sequence of a heterologous polypeptide (“added amino acids”) that is fused to a protein is encoded by an organism other than the organism from which the protein is derived, is not known to be naturally encoded by any organism, or is encoded by a gene other than the wild-type gene that encodes an endogenous version of the protein. 
     As used herein the term “signal peptide” refers to a short (e.g., 5-30 or 10-60 amino acids long) stretch of amino acids at the N-terminus of a protein that directs the transport of the protein. In various embodiments, the signal peptide is cleaved off during the post-translational modification of a protein by a cell. Signal peptides may also be referred to as “targeting signals,” “leader sequences,” “signal sequences,” “transit peptides,” or “localization signals.” In instances where a signal peptide is not defined for a ligand-binding protein discussed herein, the signal peptide may optionally be considered to be, e.g., the first 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids from the N-terminus of the translated protein (compared to a protein that has not had the signal peptide removed, e.g., compared to a naturally occurring protein). 
     In some embodiments, the ligand-binding protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 1-10, 1-15, 1-20, 5-15, 5-20, 10-25, 10-50, 20-50, 25-75, 25-100 or more mutations compared to a naturally occurring protein while retaining at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5%, or about 100% of the activity of the naturally occurring protein. Mutations include but are not limited to substitutions, insertions, and deletions. Non-limiting examples of ligand-binding proteins may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 1-10, 1-15, 1-20, 5-15, 5-20, 10-25, 10-50, 20-50, 25-75, 25-100, or more substitution mutations compared to a naturally occurring protein while retaining at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%6, or 99.5%, or about 100% of the activity of the naturally occurring protein. In embodiments, at least one amino acid of the ligand-binding protein has been substituted with a cysteine. Alternatively or in addition, a ligand-binding protein may include one or more mutations that remove a cysteine, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more substitutions or deletions of a cysteine compared to a naturally occurring protein. 
     Alternatively, the ligand-binding protein is not a mutant. For example, a reporter group is fused to the N-terminus or the C-terminus of the ligand-binding protein. 
     In some embodiments, the reporter group is conjugated to an amino acid that is no more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids from the N-terminus or the C-terminus of the ligand-binding protein. In some embodiments, the reporter group is conjugated to an amino acid that is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids from the N-terminus or the C-terminus of the ligand-binding protein. In some embodiments, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids (including or not including the signal peptide) have been deleted (e.g. are absent) from the N-terminus of the protein compared to its naturally occurring counterpart. In some embodiments, less than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids (including or not including the signal peptide) have been deleted (e.g. are absent) from the N-terminus of the protein compared to its naturally occurring counterpart. In some embodiments, about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids have been deleted (e.g. are absent) from the C-terminus of the protein compared to its naturally occurring counterpart. In some embodiments, less than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids have been deleted (e.g. are absent) from the C-terminus of the protein compared to its naturally occurring counterpart. 
     In various embodiments, a ligand-binding protein may comprise a stretch of amino acids (e.g., the entire length of the ligand-binding protein or a portion comprising at least about 50, 100, 200, 250, 300, 350, 400, or 450 amino acids) in a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, or 99.5% identical to an amino acid sequence of a naturally occurring protein. 
     In some embodiments, the mutations are conservative, and the present subject matter includes many ligand-binding proteins in which the only mutations are substitution mutations. In non-limiting examples, a ligand-binding protein has no deletions or insertions compared to a naturally occurring protein (e.g., a naturally occurring counterpart). In non-limiting examples, the ligand-binding protein does not comprise a deletion or insertion compared to its naturally occurring counterpart. Alternatively, a ligand-binding protein may have (i) less than about 5, 4, 3, 2, or 1 inserted amino acids, and/or (ii) less than about 5, 4, 3, 2, or 1 deleted amino acids compared to a naturally occurring protein. 
     In various embodiments, a naturally occurring protein to which a ligand-binding protein is compared or has been derived (e.g., by mutation, fusion, or other modification) from a prokaryotic ligand-binding protein such as a bacterial ligand-binding protein. For example, the prokaryotic ligand-binding protein is a mutant, fragment, or variant of a natural (i.e., wild-type) bacterial protein. In various embodiments, the bacterial ligand-binding protein is from a thermophilic, mesophilic, or cryophilic prokaryotic microorganism (e.g., a thermophilic, mesophilic, or cryophilic bacterium). 
     A microorganism is “thermophilic” if it is capable of surviving, growing, and reproducing at temperatures between 41 and 140° C. (106 and 284 OF), inclusive. In various embodiments, a thermophilic organism has an optimal growth temperature between 41 and 140° C., or that is at least about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140° C. Many thermophiles are archaea. Thermophilic eubacteria are suggested to have been among the earliest bacteria. Thermophiles are found in various geothermally heated regions of the Earth, such as hot springs and deep sea hydrothermal vents, as well as decaying plant matter, such as peat bogs and compost. Unlike other types of microorganisms, thermophiles can survive at much hotter temperatures, whereas other bacteria would be damaged and sometimes killed if exposed to the same temperatures. Thermophiles may be classified into three groups: (1) obligate thermophiles: (2) facultative thermophiles; and (3) hyperthermophiles. Obligate thermophiles (also called extreme thermophiles) require such high temperatures for growth, whereas facultative thermophiles (also called moderate thermophiles) can thrive at high temperatures, but also at lower temperatures (e.g. below 50° C.). Hyperthermophiles are particularly extreme thermophiles for which the optimal temperatures are above 80° C. Some microorganisms can live at temperatures higher than 100° C. at large depths in the ocean where water does not boil because of high pressure. Many hyperthermophiles are also able to withstand other environmental extremes such as high acidity or radiation levels. A compound (e.g., a protein or biosensor) is “thermotolerant” if it is capable of surviving exposure to temperatures above 41° C. For example, in some embodiments a thermotolerant biosensor retains its function and does not become denatured when exposed to a temperature of about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140° C. for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more minutes. In some embodiments, the thermotolerant compound survives exposure to 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, or 140° C. under pressure. 
     A microorganism is “mesophilic” if it is capable of surviving, growing, and reproducing at temperatures between 20 and 40° C. (68 and 104° F.), inclusive. “Psychrophiles” or “cryophiles” are microorganisms that are capable of growth and reproduction in cold temperatures. In various embodiments, a psychrophile is capable of growth and reproduction at a temperature of 10° C. or less, e.g., between −20° C. and +10° C. 
     In some embodiments, the microbial protein is produced by a bacterial microorganism, an archaean microorganism, an algal microorganism, a protozoan microorganism, or a fungal microorganism. In non-limiting examples, the microbial protein is produced by a Gram-positive bacterium or a Gram-negative bacterium. In various embodiments, a biosensor comprises a modified (e.g., mutated, fused, and/or conjugated) periplasmic binding protein or a cytoplasmic binding protein. 
     Aspects of the present subject matter provide a ligand-binding protein with a mutation that alters the interaction of the ligand-binding protein with a ligand (i.e. bicarbonate or Ca 2+ ). For example, the ligand-binding protein comprises a mutation that alters the interaction of the ligand-binding protein with the ligand compared to a naturally occurring counterpart. In some embodiments, the ligand-binding protein comprises a mutation that alters the interaction of an amino acid of the ligand-binding protein with a water molecule compared to a naturally occurring counterpart. 
     In some embodiments, the ligand-binding protein does not comprise a signal peptide. For example, the signal peptide (e.g., that is present in a naturally occurring counterpart) may be replaced with a methionine. 
     Exemplary implementations relate to a ligand such as bicarbonate or Ca 2+ , wherein the ligand-binding protein comprises a bicarbonate-binding protein or a calcium-binding protein. For example, the ligand-binding protein may comprise a mutant of, a fragment of, or a fusion protein comprising a microbial bicarbonate-binding protein. In embodiments, the ligand-binding protein is not a mutant or fragment to which a heterologous polypeptide has been attached or added. In some embodiments, the ligand-binding protein has an affinity (K d ) for a ligand within the concentration range of the ligand in a subject. In certain embodiments, the ligand-binding protein has an affinity (K d ) for ligand in the range of about 0.01 mM to about 50 mM, about 0.01 mM to about 25 mM, about 0.01 mM to about 0.1 mM, about 0.01 mM to about 0.5 mM, about 0.1 mM to about 2 mM, about 0.2 mM to about 2 mM, about 0.3 mM to about 2 mM, about 0.4 mM to about 2 mM, about 0.5 mM to about 2 mM, about 0.6 mM to about 2 mM, about 0.7 mM to about 2 mM, about 0.8 mM to about 2 mM, about 0.9 mM to about 2 mM, about 1 mM to about 2 mM, about 1.25 mM to about 2 mM, about 1.5 mM to about 2 mM, about 0.01 mM to about 10 mM, about 0.01 mM to about 5 mM, about 0.1 mM to about 50 mM, about 0.1 mM to about 25 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 5 mM, about 1 mM to about 50 mM, about 0.5 mM to about 10 mM, about 0.1 mM to about 15 mM, about 1 mM to about 25 mM, about 1 mM to about 10 mM, about 1 mM to about 5 mM, about 0.5 mM to about 1 mM, about 2 mM to about 4 mM, about 15 mM to about 35 mM, about 20 mM to about 30 mM, about 100 mM to about 150 mM, about 100 mM to about 200 mM, about 150 mM to about 250 mM, about 250 mM to about 500 mM, about 500 mM to about 750 mM, or about 750 mM to about 1000 mM. In various embodiments, the biosensor is capable of detecting ligand when ligand is present at a concentration of at least about 0.001 mM, 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, or 1000 mM. The ratiometric reagentless biosensors produce precise measurements over an extended concentration ranges, as noted above, as well as in sample volumes of less than about, e.g., 10 μl, 9 μl, 8 μl, 7 μl, 6 μl, 5 μl, 4 μl, 3 μl, 2 μl, or 1 μl. In some embodiments, the volume of sample that is applied to a biosensor or a device comprising a biosensor is less than 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 150, 300, 500, or 1000 μl. In some embodiments, the volume is about 0.1 μl to about 1000 μl, about 0.1 μl to about 100 μl, about 1 μl to about 1000 μl, about 1 μl to about 10 μl, about 1 μl to about 100 μl, about 1 μl to about 50 μl, about 10 μl to about 50 μl, or about 5 μl to about 50 μl. In some embodiments, the ligand-binding protein comprises a mutation that alters (e.g., increases or decreases) the interaction of the mutant with bound ligand compared to a naturally occurring protein (e.g., a microbial bicarbonate-binding protein). In non-limiting examples, the ligand-binding protein comprises a mutation that alters (e.g., increases or decreases) the mutant&#39;s affinity and/or specificity for ligand compared to an unmutated ligand-binding protein (e.g., a microbial bicarbonate-binding protein). In non-limiting examples, the mutant&#39;s K d  for the ligand is at least 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mM higher or lower for bicarbonate and/or Ca 2+  compared to the unmutated ligand-binding protein. 
     In certain embodiments, the ligand-binding protein comprises a mutation that alters the interaction between the protein and bound ligand, a mutation that alters the equilibrium between the open and closed states of the ligand-binding protein, a mutation that alters the interaction between the ligand-binding protein and a reporter group (such as a fluorescent conjugate, e.g., the interaction with a carbonyl group or a naphthalene ring of a prodan-derived fluorophore such as Acrylodan or Badan), and/or a mutation that impacts indirect interactions that alter the geometry of the ligand binding site. In various embodiments, the mutation does not reduce, or negligibly impacts, the thermostability of the ligand-binding protein. In some embodiments, the mutation alters the thermostability of the ligand-binding protein by less than about 1, 2, 3, 4, 5, or 10° C. 
     In some embodiments, the interaction of a bicarbonate-binding protein with a portion of bicarbonate selected from an —OH group, an oxygen atom (e.g., —O—), or a —C(═O)O −  group, or any combination thereof, is different than that of a naturally occurring counterpart of the bicarbonate-binding protein. 
     In various embodiments, the ligand-binding protein is purified. In some embodiments, the naturally occurring counterpart of the ligand-binding protein is from a Gram-positive bacterium or a Gram-negative bacterium. 
     In some embodiments, the ligand-binding protein comprises or comprises a mutant of a microbial calcium-bicarbonate binding protein. 
     The present subject matter provides a ligand-binding protein that is or is a mutant of: a  Synechocystis  sp. (e.g., S. sp. PCC6803) bicarbonate-binding protein, a  Thermosynechococcus  sp. (e.g.,  T. vulcanus, T. elongatus , or  T. elongatus  BP-1) bicarbonate-binding protein, a  Chroococcidiopsis  sp. (e.g.,  C. thermalis, C. gigantea, C. cubana , or  C. codiicola ) bicarbonate-binding protein, a  Calothrix  sp. (e.g.,  C. aberrans, C. adscencens, C aeruginea, C. aficana, C. allorgei, C. australiensis, C. hailevi, C. bharadwajae, C. borealis, C. braunii, C. breviarticulata, C. calida, C. castellii, C. capitularis, C. cavernarum Copeland, C. charicola, C. clavata, C. clavatoides, C. codicola, C. columbiana, C. compacta, C. confervicola, C. contarenii, C. coriacea, C crustacea, C. cylindrica, C. desertica, C. elsteri, C. epiphytica, C. evanescens, C. estonica, C. fasciculata, C. feldmannii, C. flahaultii, C. floccosa, C. fritschii, C. fuellebornii, C. fisca, C. fuisco - violacea, C. geilterii, C. geitonos, C ghosei. C gigas, C gloeocola, C. goetzei. C hunanica, C. inaequabilis, C. inserta, C javanica, C. karnatakensis, C. kawraiskyi, C. kossinskajae, C. kuntzei, C linearis, C minima, C. nidulans, C. parasitica, C. parietina, C. parva, C. pilosa, C. prolifera, C. pulvinata, C. rectangularis, C. reptans, C. rodriguezii, C. santapaui, C. scopulorum, C. scytonemicola, C. simplex, C. simulans. C stagnalis, C. subantarctica, C. subsimplex, C. tenella. C thermalis. C. lurfosa, C. viguieri, C. vivipara, C. violacea, C. weberi, C wembacrensis, C. aeruginosa, C. aestuarii, C. antarctica, C. atricha, C. bossei, C. brevissima, C. clausa, C conica, C. dnieprensis, C elenkinii. C fonticola, C. galpinti, C gelatinosa, C. gracilis, C intricata, C. litoralis, C. marchica, C. nodulosa, C. obtusa, C. rhizosoleniae , or  C. schweickertii ) bicarbonate-binding protein, a mutant of a  Anabaena  sp. (e.g.,  A. aequalis, A. affinis, A. angstumalis angstumalis, A. angstumalis marchita. A. aphanizomendoides, A. azollae, A. bornettana, A. catenula, A. cedrorum, A. circinalis, A. confervoides, A. constricta, A. cyanobacterium, A. cycadeae, A. cylindrica, A. echinispora, A. felisii, A. flos - aquae flos - aquae. A. flos - aquae minor, A. flos - aquae treleasei. A. helicoidea, A. inaequalis, A. lapponica, A. laxa, A. lemmermannii, A. levanderi. A. limnetica, A. macrospora macrospora, A. macrospora robusta, A. monticulosa, A. nosloc, A. oscillarioides, A. planclonica, A. raciborskii, A. scheremetievi, A. sphaerica, A. spiroides crassa, A. spiroides spiroides, A. subcylindrica, A. torulosa, A. unispora, A. variabilis, A. verrucosa, A. viguieri, A. wisconsinense , or  A. zierlingii ) bicarbonate-binding protein, or a  Chamaesiphon  sp. (e.g.,  C. africanus, C. amethystinus, C. britannicus, C. carpalicus, C. confervicola, C. cylindricus, C. cylindrosporus, C. halophilus, C. incrustans, C. investiens, C. jaoi, C. komarekii, C. longus, C. macer, C. major, C. minimus, C. minutus, C. portoricensis, C. rostafinskii, C. sideriphilus, C. tibeticus, C. aggregatus, C. fallax, C. fiiscus, C. geitleri, C. mollis, C. niger, C. ocobyrsiodes, C. polonicus, C. polymorphus, C. starmachii, C. stratosus , or  C. subglobosus ) bicarbonate-binding protein. 
     In various embodiments, a biosensor comprises a ligand-binding protein that is or is a mutant of: a bicarbonate-binding protein from  Synechocystis  sp. (synBicarbBP1; SEQ ID NO: 1, 15, or 75); a bicarbonate-binding protein from  Thermosyneochococcus elongatus  (teBicarbBP2; SEQ ID NO: 2, 16, or 76): a bicarbonate-binding protein from  Chroococcidiopsis thermalis  (ctBicarbBP3; SEQ ID NO: 3, 17, or 77); a bicarbonate-binding protein from  Calothrix  sp. (calBicarbBP4; SEQ ID NO: 4, 18, or 78); a bicarbonate-binding protein from  Anabaena variabilis  (avBicarbBP5; SEQ ID NO: 5, 19, or 79); or a bicarbonate-binding protein from  Chamaesiphon minutus  (cmBicarbBP6; SEQ ID NO: 6, 20, or 80). 
     Aspects of the present subject matter include a ligand-binding protein that is or is a mutant of a protein listed in Table 1, e.g., the protein numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 in Table 1. 
     In some embodiments, the ligand-binding protein comprises or comprises a mutant of a microbial iron-binding protein. 
     The present subject matter provides a ligand-binding protein that is or is a mutant of: a  Mannheimia  sp. (e.g.,  M. caviae, M. glucosida, M. granulomatis, M. haemolytica, M. ruminalis , or  M. varigena ) bicarbonate and iron binding protein, an  Exiguobacterium  sp. (e.g.  E. acetylicum , E. aestuari,  E. alkaliphilum, E antarcticum. E. aquaticum, E. artemiae, E. aurantiacum, E. enclense, E. indicum, E. marinum, E mexicanum. E, oxidotolerans, E. profundum, E. sibiricum, E. soli , or  E. undae ) bicarbonate and iron binding protein, a  Thermosynechococcus  sp. (e.g.,  T. vulcanus. T. elongatus , or  T. elongatus  BP-1) bicarbonate and iron binding protein, a Candidatus Nitrospira sp. (e.g.,  Candidatus Nitrospira defluvii, Candidatus Nitrospira nitrificans, Candidatus Nitrospira nitrosa, Candidatus Nitrospira inopinata, Candidatus Magnetobacterium casensis, Candidatus Magnetobacterium bavaricum, Candidatus Magnetoovum chiemensis ) bicarbonate and iron binding protein, a  Thermus  sp. (e.g.,  T. caldophilus, T. eggertssonii, T. kawarayensis, T. murrieta, T. nonproteolyvticus, T. parvatiensis, T. rehai, T. yunnanensis, T. amyloliquefaciens, T. antranikianii, T. aquaticus, T. arciformis, T. brockianus, T. caliditerrae, T. chliarophilus, T. composti, T. filiformis, T. igniterrae, T. islandicus, T. oshimai, T. profundus, T. scotoductus, T. tengchongensis , or  T. thermophilus ) bicarbonate and iron binding protein, a  Meiothermus  sp. ( M. chiliarophilus, M. cerbereus, M. granaticius, M. rosaceus, M. ruber, M. rufus, M. silvanus, M. laiwanensis , or  M. timidus ) bicarbonate and iron binding protein, a  Salinibacter  sp. (e.g.,  S. ruber, S. iranicus , or  S. luteus ) bicarbonate and iron binding protein, or a  Halorubrum  sp. (e.g.,  H. aidingense, H. alkaliphilun, H. arcis, H. californiensis, H. coriense, H. distributum, H. ejinorense, H. ezzemoulense, H. kocurii, H. lacusprofindi, H. lipolyticum, H. litoreum, H. luteum, H, orientalis, H. saccharovorum, H. salsolis, H. sodomense, H. tebenquichense, H. terrestre, H. tibetense, H. trapanicum, H. vacuolatum , or  H. xinjiangense ) bicarbonate and iron binding protein. 
     In various embodiments, a biosensor comprises a ligand-binding protein that is or is a mutant of: a bicarbonate and iron binding protein from  Mannheimia haemolytica  (mhFeBP1: SEQ ID NO: 7, 21, or 81); a bicarbonate and iron binding protein from  Exiguobacterium  sp. (exiFeBP2; SEQ ID NO: 8, 22, or 82): a bicarbonate and iron binding protein from Thermosynechoccus  elongatus  (teFeBP3; SEQ ID NO: 9, 23, or 83); a bicarbonate and iron binding protein from Candidatus nitrospira (cnFeBP4; SEQ ID NO: 10, 24, or 84); a bicarbonate and iron binding protein from  Thermus thermophilus  (ttFeBP5; SEQ ID NO: 11, 25, or 85): a bicarbonate and iron binding protein from Meiothermus  silvanus  (msFeBP6: SEQ ID NO: 12, 26, or 86); a bicarbonate and iron binding protein from  Salinibacter ruber  (srFeBP7; SEQ ID NO: 13, 27, or 87); or a bicarbonate and iron binding protein from  Halorubrum lacusprofindi  (hIFeBP8; SEQ ID NO: 14, 28, or 88). 
     Aspects of the present subject matter include a ligand-binding protein that is or is a mutant of a protein listed in Table 2, e.g., the protein numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, or 523 in Table 2. 
     Aspects of the present subject matter also include a ligand-binding protein that is or is a mutant of a protein listed in Table 3, e.g., the protein numbered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, or 71 in Table 3. 
     With regard to a defined polypeptide, % identity figures higher or lower than those provided herein will encompass various embodiments. Thus, where applicable, in light of a minimum % identity figure, a polypeptide may comprise an amino acid sequence which is at least 60%, 65%, 70%, 75%, 76%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the reference SEQ ID NO or to each of the reference SEQ ID NOs. In embodiments, the polypeptide comprises an amino acid sequence that is 100% identical to the reference SEQ ID NO. Where applicable, in light of a maximum % identity to a reference sequence, a polypeptide may comprise an amino acid sequence which is less than 75%, 70%, 65%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%6, 32%, 31%, 30%, 29%6, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 210% 20%, 19%, 18%, 17%, 16%, or 15% identical to the reference SEQ ID NO or to each of the reference SEQ ID NOs. In certain embodiments, a polypeptide comprises amino acids in a sequence that is preferably at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%6, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% and less than about 75%, 70%, 65%, 60%, 55%, 50%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, or 30% identical to the reference SEQ ID NO or to each of the reference SEQ ID NOs. In certain embodiments, a polypeptide comprises amino acids in a sequence that is between about 10% and about 60%, 11% and about 60%, 12% and about 60%, 13% and about 60%, 14% and about 60%, 15% and about 60%, 16% and about 60%, 17% and about 60%, 18% and about 60%, 19% and about 60%, 20% and about 60%, 21% and about 60%6, 22% and about 60%, 23% and about 60%, 24% and about 60%, 25% and about 60%, 26% and about 60%, 27% and about 60%, 28% and about 60%, 29% and about 60%, 30% and about 60%, about 25% and about 100%, about 25% and about 95%, about 25% and about 85%, about 25% and about 75%, about 25% and about 70%, about 25% and about 65%, 60%, about 25% and about 55%, about 25% and about 50%, about 25% and about 45%, about 25% and about 44%, about 25% and about 43%, about 25% and about 42%, about 25% and about 41%, about 25% and about 40%, about 25% and about 39%, about 25% and about 38%, about 25% and about 37%, about 25% and about 36%, about 25% and about 35%, about 25% and about 34%, about 25% and about 33%, about 25% and about 32%6, about 25% and about 31%, or about 25% and about 30% identical to the reference SEQ ID NO or to each of the reference SEQ ID NOs. Non-limiting examples of reference proteins and amino acid sequences disclosed herein include:
         (i) a bicarbonate-binding protein from  Stnechocystis  sp. (synBicarbBP1; genome, NC_017052, protein YP_005410477.1; SEQ ID NO: 1);   (ii) a bicarbonate-binding protein from  Thermosynechococcus elongatus  (teBicarbBP2; genome, NC_004113, protein NP_682790.1; SEQ ID NO: 2);   (iii) a bicarbonate-binding protein from Chroococcidiopsis  thermalis  (ctBicarbBP3; genome, NC_019695, protein YP_007090308.1; SEQ ID NO: 3);   (iv) a bicarbonate-binding protein from  Calothrix  sp. (calBicarbBP4; genome, NC_019751, protein YP_007137061.1; SEQ ID NO: 4);   (v) a bicarbonate-binding protein from  Anabaena variabilis  (avBicarbBP5; genome, NC_007413, protein YP_321546.1; SEQ ID NO: 5);   (vi) a bicarbonate-binding protein from Chamaesiphon  minutus  (cmBicarbBP6; genome, NC_019697, protein YP_007099445.1; SEQ ID NO: 6);   (vii) a bicarbonate and iron binding protein from  Mannheimia haemolytica  (mhFeBP1; genome, NC_0121082, protein, YP_007884192.1; SEQ ID NO: 7);   (viii) a bicarbonate and iron binding protein from  Exiguobacterium  sp. (exiFeBP2; genome, NC_012673, protein, YP_002886303.1; SEQ ID NO: 8);   (ix) a bicarbonate and iron binding protein from  Thermosynechoccus elongatus  (teFeBP3; genome, NC_004113, protein, NP_681303.1; SEQ ID NO: 9);   (x) a bicarbonate and iron binding protein from  Candidatus nitrospira  (cnFeBP4; genome, NC_014355, protein, YP_003796723.1; SEQ ID NO: 10);   (xi) a bicarbonate and iron binding protein from  Thermus thermophilus  (ttFeBP5; genome, NC_006461, protein, YP_144894.1; SEQ ID NO: 11);   (xii) a bicarbonate and iron binding protein from  Meiothermus silvanus  (msFeBP6; genome, NC_014212, protein, YP_003686074.1; SEQ ID NO: 12);   (xiii) a bicarbonate and iron binding protein from  Salinibacter ruber  (srFeBP7; genome, NC_014032, protein, YP_003572493.1; SEQ ID NO: 13); and   (xiv) a bicarbonate and iron binding protein from  Halorubrum lacusprofundi  (hlFeBP8; genome, NC_012029, protein, YP_002564837.1; SEQ ID NO: 14).       

     In some embodiments, the ligand-binding protein comprises an amino acid sequence with at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, or 100% identity to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more ligand-binding proteins disclosed herein. 
     The ligand-binding proteins disclosed herein may optionally be fused (e.g., at their N-terminal and/or C-terminal ends) to a motif comprising a stretch of amino acids that facilitates the isolation or other manipulation such as conjugation to a moiety or immobilization on a substrate such as a plastic, a cellulose product such as paper, polymer, metal, noble metal, semi-conductor, or quantum dot (e.g., a fluorescent quantum dot). A non-limiting example of such a stretch of amino acids has the sequence: GGSHHHHHH (SEQ ID NO: 89). This motif is not required for, is not believed to influence or affect ligand-binding activity or signal transduction, and may be omitted from any ligand-binding protein or biosensor disclosed herein. Additionally, for every sequence disclosed herein that includes GGSHHHHHH (SEQ ID NO: 89), a corresponding sequence that is identical except that it lacks GGSHHHHHH (SEQ ID NO: 89) is also provided and intended to be disclosed. For example, each of SEQ ID NOs: 1-74 (and the non-limiting examples of other proteins used in the experiments disclosed herein) comprises this motif (SEQ ID NO: 89). Alternatively or in addition, a ligand-binding protein may be fused to a heterologous polypeptide or “added amino acids” that facilitates the attachment thereof to a surface, such as the surface of a device. 
     In some embodiments, a polypeptide comprises 1, 2, 3, 4, 5, or more substitutions or deletions of a cysteine compared to the naturally occurring counterpart of the polypeptide (i.e., 1, 2, 3, 4, 5, or more native cysteines have been removed), e.g., 1, 2, 3, 4, 5, or more cysteine to alanine substitutions compared to the naturally occurring counterpart of the polypeptide. In some embodiments, all of the cysteines of a polypeptide have been deleted and/or substituted compared to its natural counterpart. In some embodiments, one or more cysteines of a polypeptide have been substituted with an alanine, a serine, or a threonine. 
     In embodiments, the amino acid sequence of a protein comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mutations compared to its naturally occurring counterpart. In some embodiments, less than 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 of the mutations is a deletion or insertion of 1, 2, 3, 4, or 5 or no more than 1, 2, 3, 4, or 5 amino acids. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more of the mutations is a substitution mutation. In certain embodiments, every mutation to a protein compared to its naturally occurring counterpart is a substitution mutation. In various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more or all of the mutations to a protein compared to its naturally occurring counterpart is a conservative substitution mutation. 
     In various embodiments, a polypeptide does not have any insertion or deletion compared to its natural counterpart, other than (optionally) the removal of the signal peptide and/or the fusion of compounds such as another polypeptide at the N-terminus or C-terminus thereof. 
     Ligand-Binding Proteins Comprising a Primary Complementary Surface (PCS) The following BLAST parameters are used to identify sequence homologues of a bicarbonate-binding protein (such as synBicarbBP1, mhFeBP1, or ttFeBP5): (1) Expect threshold is 10.0; (2) Gap cost is Existence: 11 and Extension: 1: (3) The Matrix employed is BLOSUM62; (4) The filter for low complexity regions is “on.” Such an alignment may be generated using the ProteinHunter program. The ProteinHunter package always executes BLAST searches, with the following command 
     “blastall -p blastp -m 8-b 50000 -d % s -i&lt;INPUT FILE&gt; -o&lt;OUTPUT FILE&gt;” 
     where &lt;INPUT FILE&gt; and &lt;OUTPUT FILE&gt; specify the input and output files, respectively for a given calculation. This command executes the BLAST alignment program for protein sequences with default parameters, intrinsically set by the program. The BLAST program version is 2.2.24. 
     Sequence homologues of synBicarbBP1, mhFeBP1, or ttFeBP5 identified using BLAST may be aligned with synBicarbBP1, mhFeBP1, or ttFeBP5 using ClustalW to identify homologues that share a PCS with synBicarbBP1, mhFeBP1, or ttFeBP5 as discussed below. 
     Aspects of the present subject matter provide ligand-binding proteins that share a PCS with a bicarbonate-binding protein disclosed herein. 
     In embodiments, the PCS comprises at least about 3, 4, 5, 6, 7, or 8 amino acid positions used to identify a bicarbonate-binding protein. For example, the PCS of synBicarbBP1 may comprise positions 20, 49, 71, 102, 142, 148, 220, and 221, wherein each position is counted as in SEQ ID NO: 15 or 75. In various embodiments, a protein shares a PCS with synBicarbBP1 if the amino acid sequence of the protein has 
     (i) E at the position that aligns with position 20 of synBicarbBP1; 
     (ii) W at the position that aligns with position 49 of synBicarbBP1; 
     (iii) Q at the position that aligns with position 71 of synBicarbBP1; 
     (iv) N at the position that aligns with position 102 of synBicarbBP1; 
     (v) T at the position that aligns with position 142 of synBicarbBP1; 
     (vi) Q at the position that aligns with position 148 of synBicarbBP1; 
     (vii) E at the position that aligns with position 220 of synBicarbBP1; and 
     (viii) E at the position that aligns with position 221 of synBicarbBP1, 
     wherein the alignment between synBicarbBP1 (SEQ ID NO: 15 or 75) and the protein is constructed using the ClustalW alignment program. 
     In embodiments, the PCS comprises at least about 3, 4, or 5 amino acid positions used to identify a bicarbonate-binding protein. For example, the PCS of mhFeBP1 may comprise positions 11, 102, 143, 199, and 200 wherein each position is counted as in SEQ ID NO: 21 or 81. In various embodiments, a protein shares a PCS with mhFeBP1 if the amino acid sequence of the protein has 
     (i) R at the position that aligns with position 11 of mhFeBP1; 
     (ii) R at the position that aligns with position 102 of mhFeBP1; 
     (iii) Y at the position that aligns with position 143 of mhFeBP1; 
     (iv) Y at the position that aligns with position 199 of mhFeBP1; and 
     (v) Y at the position that aligns with position 200 of mhFeBP1, 
     wherein the alignment between mhFeBP1 (SEQ ID NO: 21 or 81) and the protein is constructed using the ClustalW alignment program. 
     In embodiments, the PCS comprises at least about 3, 4, or 5 amino acid positions used to identify a bicarbonate-binding protein. For example, the PCS of ttFeBP5 may comprise positions 11, 101, 143, 200, and 201 wherein each position is counted as in SEQ ID NO: 25 or 85. In various embodiments, a protein shares a PCS with ttFeBP5 if the amino acid sequence of the protein has 
     (i) R at the position that aligns with position 11 of ttFeBP5; 
     (ii) R at the position that aligns with position 101 of ttFeBP5; 
     (iii) Y at the position that aligns with position 143 of ttFeBP5; 
     (iv) Y at the position that aligns with position 200 of ttFeBP5; and 
     (v) Y at the position that aligns with position 201 of ttFeBP5, 
     wherein the alignment between ttFeBP5 (SEQ ID NO: 25 or 85) and the protein is constructed using the ClustalW alignment program. 
     The ProteinHunter package always executes multiple sequence alignments with the following command 
     “clustalw -infile=&lt;INPUT FILE&gt; -outfile=&lt;OUTPUTFILE&gt; -align -quiet” 
     This command executes the CLUSTALW multi-sequence alignment program for protein sequences. There are no user-specified parameter settings that alter the alignment behavior of the program. The CLUSTALW program version is 2.1. 
     For convenience and depending on context, a position that aligns with a stated position of synBicarbBP1, mhFeBP1, or ttFeBP5 may be referred to herein as “equivalent” to the stated position. 
     Exemplary Ligand-Binding Proteins 
     Various biosensors provided herein comprise ligand-binding proteins, such as ligand-binding proteins that have altered amino acid sequences compared to their naturally occurring counterparts. In embodiments, such proteins are conjugated to reporter groups. 
     In various embodiments, the C α  root-mean-square deviation (RMSD) between the backbone of the ligand-binding polypeptide and synBicarbBP1, teBicarbBP2, ctBicarbBP3, calBicarbBP4, avBicarbBP5, cmBicarbBP6, mhFeBP1, exiFeBP2, teFeBP3, cnFeBP4, ttFeBP5, msFeBP6, srFeBP7, and/or hlFeBP8 is, e.g., between about 0-3 Å, 0-1 Å, 0-1.5 Å, 0-2 Å, 0.1-3 Å, 0.5-1 Å, 0.5-1.5 Å, or 0.5-2 Å, or less than about 0.1 Å, 0.2 Å, 0.3 Å, 0.4 Å, 0.5 Å, 0.6 Å, 0.7 Å, 0.8 Å, 0.9 Å, 1.0 Å, 1.5 Å, 1.6 Å, 1.7 Å, 1.8 Å, 1.9 Å, 2.0, 2.5 Å, or 3 Å. In some embodiments, the Ca RMSD between the N-terminal domain (i.e., the portion of the protein at the N-terminal side of the binding domain hinge) backbone of the ligand-binding polypeptide and the corresponding domain of synBicarbBP1, teBicarbBP2, ctBicarbBP3, calBicarbBP4, avBicarbBP5, cmBicarbBP6, mhFeBP1, exiFeBP2, teFeBP3, cnFeBP4, ttFeBP5. msFeBP6, srFeBP7. and/or hlFeBP8 is, e.g., between about 0-3 Å, 0-1 Å, 0-1.5 Å, 0-2 Å, 0.1-3 Å, 0.5-1 Å, 0.5-1.5 Å, or 0.5-2 Å, or less than about 0.1 Å, 0.2 Å, 0.3 Å, 0.4 Å, 0.5 Å, 0.6 Å, 0.7 Å, 0.8 Å, 0.9 Å, 1.0 Å, 1.5 Å, 1.6 Å, 1.7 Å, 1.8 Å, 1.9 Å, 2.0 Å, 2.5 Å, or 3 Å. In certain embodiments, the Ca RMSD between the C-terminal domain (i.e., the portion of the protein at the C-terminal side of the binding domain hinge) backbone of the ligand-binding polypeptide and the corresponding domain of synBicarbBP1, teBicarbBP2, ctBicarbBP3, calBicarbBP4, avBicarbBP5, cmBicarbBP6, mhFeBP1, exiFeBP2, teFeBP3, cnFeBP4, ttFeBP5, msFeBP6, srFeBP7, and/or hlFeBP8 is, e.g., between about 0-3 Å, 0-1 Å, 0-1.5 Å, 0-2 Å, 0.1-3 Å, 0.5-1 Å, 0.5-1.5 Å, or 0.5-2 Å, or less than about 0.1 Å, 0.2 Å, 0.3 Å, 0.4 Å, 0.5 Å, 0.6 Å, 0.7 Å, 0.8 Å, 0.9 Å, 1.0 Å, 1.5 Å, 1.6 Å, 1.7 Å, 1.8 Å, 1.9 Å, 2.0 Å, 2.5 Å, or 3 Å. Non-limiting considerations relating to the sequence and structural differences between homologous proteins are discussed in Chothia and Lesk (1986)  The EMBO Journal,  5(4):823-826, the entire content of which is incorporated herein by reference. 
     Non-limiting examples of ligand-binding polypeptides that are useful in biosensors provided herein include synBicarbBP1, teBicarbBP2, ctBicarbBP3, calBicarbBP4, avBicarbBP5, cmBicarbBP6, mhFeBP1, exiFeBP2, teFeBP3, cnFeBP4, ttFeBP5, msFeBP6, srFeBP7, and hlFeBP8. In embodiments, a biosensor comprises a modified synBicarbBP1, teBicarbBP2, ctBicarbBP3, calBicarbBP4. avBicarbBP5, cmBicarbBP6, mhFeBP1, exiFeBP2, teFeBP3, cnFeBP4, ttFeBP5, msFeBP6, srFeBP7, or hlFeBP8 polypeptide having an amino acid substitution compared to its naturally occurring counterpart, such that the polypeptide has a cysteine at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, or 460 or any combination of 1, 2, 3, 4, or 5 thereof, wherein the position corresponds a SEQ ID NO disclosed herein for synBicarbBP1, teBicarbBP2, ctBicarbBP3, calBicarbBP4, avBicarbBP5, cmBicarbBP6, mhFeBP1, exiFeBP2, teFeBP3, cnFeBP4, ttFeBP5, msFeBP6, srFeBP7, or hlFeBP8. In embodiments, the cysteine is conjugated to a reporter group. 
     In various embodiments, the disassociation constant of the mutant ligand-binding polypeptide differs by at least about 1 μM, 5 μM, 10 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 75 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM (increase or decrease) compared to its naturally occurring counterpart. 
     The biosensors and ligand-binding proteins provided herein are robust and useful at a wide range of physical conditions, e.g., pressure, temperature, salinity, osmolality, and pH conditions. For example, biosensors and ligand-binding proteins provided herein may survive substantial periods of time after being dried or exposed to high temperatures. In some embodiments, the biosensor maintains at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more of its signal transduction activity after exposure to a temperature of about 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, or 125, or 40-125° C., for about 1, 2, 3, 4, 5, 6, 15, 30, 60, 120, 180, 240, or 360 minutes. In certain embodiments, the biosensor maintains at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more of its signal transduction activity after 1, 2, 3, 4, or 5 freeze-thaw cycles in an aqueous solution. In various embodiments, the biosensor maintains at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more of its signal transduction activity after storage at a temperature of between 20-37° C. for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, or 1-24 months in dry form. In some embodiments, the optimal functional temperature of the biosensor is between 41 and 122° C., between 20 and 40° C., or less than about 10° C. (e.g., between −20 and +10° C.). Devices, compositions, and biosensors provided herein may be stored, e.g., with or without protection from exposure to light. In some embodiments, the devices, compositions, and biosensors are stored in the dark, e.g., with protection from light. synBicarbBP1 is a non-limiting reference protein respect to bicarbonate-binding proteins and calcium-binding proteins. An alignment of synBicarbBP1 with other polypeptides is provided in  FIG. 5 . 
     In various embodiments, a ligand-binding protein (or its naturally occurring counterpart) comprises
         (a) an amino acid sequence that is preferably (i) at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, and (ii) less than about 75%, 70%, 65%, 60%, 55%, 50%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, or 35% identical to synBicarbBP1;   (b) a cysteine substitution (compared to its naturally occurring counterpart) within a stretch of at least 5, 10, or 20 amino acids having at least about 50%, 55%, 610, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 18 of synBicarbBP1;   (c) a stretch of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 18 of synBicarbBP1;   (d) a stretch of amino acids in the sequence KLGX 1 IX 2 IX 3 EX 4 AP (where X 1  is any amino acid, or where X 1  is F or Y; where X 2  is any amino acid, or where X 2  is P or A; where X 3  is any amino acid, or where X 3  is V or A; and where X 4  is any amino acid, or where X 4  is S or A) (SEQ ID NO: 155);   (e) a cysteine substitution (compared to its naturally occurring counterpart) within a stretch of at least 5, 10, or 20 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80% 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 70 of synBicarbBP1;   (f) a stretch of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 70 of synBicarbBP1;   (g) a stretch of amino acids in the sequence DGGQXQMPMP (where X is any amino acid, or where X is W or Y) (SEQ ID NO: 156);   (h) a cysteine substitution (compared to its naturally occurring counterpart) within a stretch of at least 5, 10, or 20 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 103 of synBicarbBP1;   (i) a stretch of at least 4, 5, 6, 7. 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 103 of synBicarbBP1;   (j) a stretch of amino acids in the sequence GNGIA (SEQ ID NO: 157);   (k) a cysteine substitution (compared to its naturally occurring counterpart) within a stretch of at least 5, 10, or 20 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 144 of synBicarbBP1;   (l) a stretch of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 144 of synBicarbBP1;   (m) a stretch of amino acids in the sequence TFX 1 X 2 VNQD (where X 1  is any amino acid, or where X 1  is P or A; and where X 2  is any amino acid, or where X 2  is N, H, Q, or R) (SEQ ID NO: 158);   (n) a cysteine substitution (compared to its naturally occurring counterpart) within a stretch of at least 5, 10, or 20 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 220 of synBicarbBP1;   (o) a stretch of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 220 of synBicarbBP1;   (p) a stretch of amino acids in the sequence HPEEY (SEQ ID NO: 159);   (q) no more than 1, 2. 3, 4, 5, 6, 7, 8, 9, 10, or 15 deleted or inserted amino acids compared to synBicarbBP1, not including amino acids added to the N-terminus or C-terminus of the polypeptide compared to its natural counterpart, and including or not including the signal peptide of the natural counterpart of the polypeptide;   (r) at least 10, 11, 12, 13, or 14, or exactly 10, 11, 12, 13, or 14 α-helices; and/or   (s) at 10, 11, 12, 13, or 14 β-strands or exactly 10, 11, 12, 13, or 14 β-strands.       

     In embodiments, two or more or each of features (b)-(p) above occurs in the polypeptide in the order listed above as the amino acid sequence of the polypeptide is viewed or read from the N-terminus to the C-terminus (with additional features and/or amino acid sequences therebetween). For example, the polypeptide may have an N-terminus, followed by feature (b), (c), or (d), followed by feature (e), (f), or (g), followed by feature (h), (i), or (j), followed by feature (k), (l), or (m), followed by feature (n), (o), or (p), followed by the C-terminus. 
     As used herein when referring to the order of features in an amino acid read from the N terminus to the C-terminus, a first feature is “followed by” a second feature when the second feature occurs after the first feature in the amino acid sequence. The words “followed by” do not require that the second feature immediately follow or be close to the first feature. For example, the N-terminus is followed by the C-terminus. 
     The features listed above are not limiting and may be combined with any other relevant features disclosed herein, including those listed below. 
     In some embodiments the polypeptide comprises the following sequence: 
                    XXXXMXXXSRRKFLLTAGAXAXGAXFLKGCAGNPPXPXXXXXXXQXXXXX               AXXLSXEXXPETTX!KLG%IPIVESAPLIIAKEKGFFAKYG$TXV#VSKQ               ASWGSARDNVEIGSAGGG!DGGQWQMPMPHLITEGIITKGNXKIPMYVLA               QLXTQGNGIA!AXXHXGKGXXLXXXXXAXYXXGXXKXXGXPFKAAXTFPX               VNQDFWIRYWLAAGGI#P#XD!XLLAVPAA#TVAXMRTGTMDAFSTGDPW               PXRIVX#XXKIG%$AXLTA#IWPXHPEEYLAXRA#WVDKHPKATKALLKG               !MEAQQWXD#XXNRXEXAXILXGRXYF#XPXXXILXXP%XGXYX$GDGRX               XXDDXXXAXLYWKDXXGNX!SYPYKSHDLWFLTESVRWGFLPXDXLXXXX               XXAXXXIXKVNREDLWXEAAK#LGIAAA#IPTSTSRG!ETFFDGXKF#PE               NPXAYLXSLKIKKXXX            
wherein each
         X is, individually, any amino acid or is absent,   i is, individually, I or V,   $ is, individually, L or M,   % is, individually, F or Y, and   # is, individually, N, D, Q, or E.       

     In a non-limiting example, the ligand-binding protein comprises an N-terminal domain and a C-terminal domain connected by a flexible hinge, with the ligand-binding site (the ligand binding domain) located in the cleft between the N-terminal and the C-terminal domain. 
     In some embodiments, the ligand-binding protein comprises, from the N-terminus to the C-terminus, a first β-strand (β1), followed by a first α-helix (α1), followed by a second β-strand (β2), followed by a second α-helix (α2), followed by a third β-strand (β3), followed by a third α-helix (α3), followed by a fourth β-strand (β4), followed by a fifth β-strand (β5), followed by a fourth α-helix (α4), followed by a sixth β-strand (β6), followed by a fifth α-helix (α5), followed by a sixth α-helix (α6), followed by a seventh β-strand (β7), followed by a seventh α-helix (α7), followed by an eighth β-strand (β8), followed by a ninth β-strand (β9), followed by an eighth α-helix (α8), followed by a ninth α-helix (α9), followed by a tenth β-strand (β10), followed by a tenth α-helix (α10), followed by an eleventh β-strand (β11). followed by a twelfth β-strand (β12), followed by a thirteenth β-strand (β13), followed by an eleventh α-helix (α11), followed by a twelfth α-helix (α12), followed by a thirteenth α-helix (α13), followed by a fourteenth β-strand (β14), followed by a fourteenth α-helix (α14). In some embodiments, the polypeptide comprises (i) 1, 2, or 3 amino acid substitutions between β1 and α1: (ii) 1, 2, or 3 amino acid substitutions between α1 and β2; (iii) 1, 2, or 3 amino acid substitutions between β2 and α2; (iv) 1, 2, or 3 amino acid substitutions between α2 and β3: (v) 1, 2, or 3 amino acid substitutions between the β3 and α3; (vi) 1, 2, or 3 amino acid substitutions between α3 and α4; (vii) 1, 2, or 3 amino acid substitutions between β4 and β5; (viii) 1, 2, or 3 amino acid substitutions between β5 and α4; (ix) 1, 2, or 3 amino acid substitutions between α4 and β6; (x) 1, 2, or 3 amino acid substitutions between β6 and α5; (xi) 1, 2, or 3 amino acid substitutions between β5 and α6; (xii) 1, 2, or 3 amino acid substitutions between α6 and β7; (xiii) 1, 2, or 3 amino acid substitutions between β7 and α7; (xiv) 1, 2, or 3 amino acid substitutions between α7 and β8; (xv) 1, 2, or 3 amino acid substitutions between β8 and β9; (xvi) 1, 2, or 3 amino acid substitutions between β9 and α8; (xvii) 1, 2, or 3 amino acid substitutions between α8 and α9; (xviii) 1, 2, or 3 amino acid substitutions between α9 and β10; (xix) 1, 2, or 3 amino acid substitutions between β10 and α10; (xx) 1, 2, or 3 amino acid substitutions between α10 and β11; (xxi) 1, 2, or 3 amino acid substitutions between β11 and β12; (xxii) 1, 2, or 3 amino acid substitutions between β12 and β13; (xxiii) 1, 2, or 3 amino acid substitutions between β13 and α11; (xxiv) 1, 2, or 3 amino acid substitutions between α11 and α12; (xxv) 1, 2, or 3 amino acid substitutions between α12 and α13; (xxvi) 1, 2, or 3 amino acid substitutions between α13 and β14; (xxvii) 1, 2, or 3 amino acid substitutions between β14 and α14; (xxviii) 1, 2, or 3 amino acid substitutions in α1, α2, α3, α4, α5, α6, α7, α8, α9, α10, α11, α12, α13, or α14; and/or (xxix) 1, 2, or 3 amino acid substitutions in β1, β2, β3, β4, β5, β6, β7, β8, β9, β10, β11, β12, β13, or β14. In some embodiments, the substitutions are conservative substitutions. In various embodiments, the polypeptide comprises a cysteine substitution within α2, α5, α7, β1, or β7, or between β1 and α1, β3 and α3, or β6 and α5. 
     The ligand-binding polypeptide may further comprise 1, 2, or more Ca 2+  binding sites. 
     Beta sheets consist of beta strands (also β-strand) connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet. A β-strand is a stretch of polypeptide chain, e.g. 3 to 20 amino acids long, with backbone in an extended conformation. 
     Alpha-helical and β-strand segments assignments are calculated from a three-dimensional protein structure as follows, and as described in C. A. F. Andersen, B. Rost, 2003 , Structural Bioinformatics,  341-363. P. E. Bourne, ed., Wiley, the entire content of which is incorporated herein by reference. First for a given residue, i, the backbone trace angle, r, is calculated, defined as the dihedral angle between the four successive C α  atom positions of residues in the linear protein sequence i, i+1, i+2, i+3. These values are calculated for all residues. Second, the residues that form backbone hydrogen bonds with each other are recorded. A hydrogen bond is scored if the distance between the backbone amide nitrogen and carbonyl oxygen of two different residues in the protein is calculated to be 2.5 Å or less, and if the calculated angle between the nitrogen, its amide proton, and the carbonyl is greater than 120°. A residue is deemed to be in an α-helix, if 35≤τ≤65, and it makes a backbone hydrogen bond with its i+4 th  neighbor in the linear amino acid sequence. It is deemed to be in a β-strand, if the absolute t value falls in the interval 120≤|τ|≤180 and if it makes at least one hydrogen bond with another residue with the same τ value range. Alpha-helical segments comprise at least four residues; β-strand residues comprise at least three residues. 
     In embodiments, a biosensor comprises a modified synBicarbBP1. In non-limiting examples, the modified synBicarbBP1 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: I16X, P17X, I18X, W49X, Q71X, H141X, T142X, F143X, P144X, N147X, T191X, and W195X, where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in synBicarbBP1 with the signal peptide replaced with a methionine (SEQ ID NO: 15 or 75). In some embodiments, the modified synBicarbBP1 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the following substitutions: I16A, I16C, I16M, I16F, I16Y, I16E, I16W, P17C, I18C, W49A, W49C, W49F, W49Y, Q71C, H141C, T142A, T142C, T142F, T142Y, T142W, T142Q, T142E, T142V, F143C, P144C, N147C, T191C, and W195C. 
     In various embodiments, the ligand-binding protein comprises a mutation that decreases bicarbonate binding compared to a naturally occurring counterpart. In non-limiting examples, the modified synBicarbBP1 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: I16X, W49X, Q71X, and T142X, where X is any amino acid, an amino acid that results in a conservative substitution, or an alanine, and where each position is counted in synBicarbBP1 with the signal peptide replaced with a methionine (SEQ ID NO: 15 or 75). In some embodiments, the modified synBicarbBP1 comprises 1 or 2 of the following substitutions: I16A, I16C, I16M, I16F, I16Y, I16E, I16W, W49A, W49C, W49C, W49F, W49Y, Q71C, T142A, T142C, T142F, T142Y, T142W, T142Q, T142E, and T142V. 
     In embodiments, a biosensor comprises a modified teBicarbBP2. In non-limiting examples, the modified teBicarbBP2 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: I12X, P13X, I14X, W45X, Q67X, H137X, T138X, F139X, P140X, N143X, T187X, W191X, C247X, and C260X, where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in teBicarbBP2 with the signal peptide replaced with a methionine (SEQ ID NO: 16 or 76). The sequence for teBicarbBP2 (SEQ ID NO: 16 or 76) comprises C247A and C260A mutations. In some embodiments, the modified teBicarbBP2 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the following substitutions: I12A, I112C, I112M, I12F, I12Y, I112E, I12W, P13C, I14C, W45A, W45C. W45F, W45Y, Q67C, H137C, T138A, T138C, T138F, T138Y, T138W, T138Q, T138E, T138V, F139C, P140C, N143C, T187C, W191C, C247A, and C260A. 
     In various embodiments, the ligand-binding protein comprises a mutation that decreases bicarbonate binding compared to a naturally occurring counterpart. In non-limiting examples, the modified teBicarbBP2 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: I12X, W45X, Q67X, and T138X, where X is any amino acid, an amino acid that results in a conservative substitution, or an alanine, and where each position is counted in teBicarbBP2 with the signal peptide replaced with a methionine (SEQ ID NO: 16 or 76). In some embodiments, the modified teBicarbBP2 comprises 1 or 2 of the following substitutions: I12A, I12C, 112M. I12F, I12Y, I12E, 112W, W45A, W45C, W45F, W45Y, Q67C, T138A, T138C, T138F, T138Y, T138W, T138Q, T138E, and T138V. 
     In embodiments, a biosensor comprises a modified ctBicarbBP3. In non-limiting examples, the modified ctBicarbBP3 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: I16X, P17X, I18X, W49X, Q71X, Y142X, T143X, F144X, A145X, N148X, T192X, and W196X, where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in ctBicarbBP3 with the signal peptide replaced with a methionine (SEQ ID NO: 17 or 77). In some embodiments, the modified ctBicarbBP3 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the following substitutions: I16A, I16C, I16M, I16F, I16Y, I16E, I16W, P17C, I18C, W49A, W49C, W49F, W49Y, Q71C, Y142C, T143A, T143C, T143F, T143Y, T143W, T143Q, T143E, T143V, F144C, A145C, N148C, T192C, and W196C. 
     In various embodiments, the ligand-binding protein comprises a mutation that decreases bicarbonate binding compared to a naturally occurring counterpart. In non-limiting examples, the modified ctBicarbBP3 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: I16X, W49X, Q71X, and T143X, where X is any amino acid, an amino acid that results in a conservative substitution, or an alanine, and where each position is counted in ctBicarbBP3 with the signal peptide replaced with a methionine (SEQ ID NO: 17 or 77). In some embodiments, the modified ctBicarbBP3 comprises 1 or 2 of the following substitutions: I16A, I16C, I16M, I16F, I16Y, I16E, I16E, W49A, W49C, W49F, W49Y, Q71C, T143A, T143C, T143F. T143Y, T143W, T143Q, T143E, and T143V. 
     In embodiments, a biosensor comprises a modified calBicarbBP4. In non-limiting examples, the modified calBicarbBP4 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: I16X, P17X, I18X, W49X, Q71X, F140X, T141X, F142X, P143X, N146X, T190X, and W194X, where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in calBicarbBP4 with the signal peptide replaced with a methionine (SEQ ID NO: 18 or 78). In some embodiments, the modified calBicarbBP4 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the following substitutions: I16A, I16C, I16M, I16F, I16Y, I16E, I16W, P17C, I18C, W49A, W49C, W49F, W49Y, Q71C, F140C, T141A, T141C, T141F, T141Y, T141W, T141Q, T141E, T141V, F142C, P143C, N146C, T190C, and W196C. 
     In various embodiments, the ligand-binding protein comprises a mutation that decreases bicarbonate binding compared to a naturally occurring counterpart. In non-limiting examples, the modified calBicarbBP4 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: I16X, W49X, Q71X, and T141X, where X is any amino acid, an amino acid that results in a conservative substitution, or an alanine, and where each position is counted in calBicarbBP4 with the signal peptide replaced with a methionine (SEQ ID NO: 18 or 78). In some embodiments, the modified calBicarbBP4 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the following substitutions: I16A, I16C, I16M, I16F, I16Y, I16E, I16W, W49A, W49C, W49F, W49Y, Q71C, T141A, T141C, T141F, T141Y, T141W, T141Q, T141E, and T141V. 
     In embodiments, a biosensor comprises a modified avBicarbBP5. In non-limiting examples, the modified avBicarbBP5 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: I16X, P17X, I18X, W49X, Q71X, C96X, F140X, T141X, F142X, P143X, N146X, T190X, and W194X, where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in avBicarbBP5 with the signal peptide replaced with a methionine (SEQ ID NO: 19 or 79). The sequence for avBicarbBP5 (SEQ ID NO: 19 or 79) comprises a C96A mutation. In some embodiments, the modified avBicarbBP5 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the following substitutions: I16A, I16C, I16M, I16F, I16Y, I16E, I16W, P17C, I18C, W49A, W49C, W49F, W49Y, Q71C, Q71D, Q71N, Q71E, Q71M, C96A, F140C, T141A, T141C, T141F, T141Y, T141W, T141Q, T141E, T141V, F142C, P143C, N146C, T190C, and W194C. 
     In various embodiments, the ligand-binding protein comprises a mutation that decreases bicarbonate binding compared to a naturally occurring counterpart. In non-limiting examples, the modified avBicarbBP5 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: I16X, W49X, Q71X, and T141X, where X is any amino acid, an amino acid that results in a conservative substitution, or an alanine, and where each position is counted in avBicarbBP5 with the signal peptide replaced with a methionine (SEQ ID NO: 19 or 79). In some embodiments, the modified avBicarbBP5 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the following substitutions: I16A, I16C, I16M, I16F, I16Y, I16E, I16W, W49A, W49C, W49F, W49Y, Q71C, T141A, T141C, T141F, T141Y, T141W, T141Q, T141E, and T141V. 
     In embodiments, a biosensor comprises a modified cmBicarbBP6. In non-limiting examples, the modified cmBicarbBP6 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: I17X, A18X, I19X, W50X, Q72X, Y142X, T143X, F144X, P145X, N148X, T192X, W196X, and C254X, where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in cmBicarbBP6 with the signal peptide replaced with a methionine (SEQ ID NO: 20 or 80). The sequence for cmBicarbBP6 (SEQ ID NO: 20 or 80) comprises a C254A mutation. In some embodiments, the modified cmBicarbBP6 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the following substitutions: I17A, I17C, I17M, I17F, I17Y, I17E, I17W, A18C, I19C, W50A, W50C, W50F, W50Y, Q72C, Y142C, T143A, T143C, T143F, T143Y, T143W, T143Q, T143E, T143V, F144C, P145C, N148C, T192C, W196C, and C254A. 
     In various embodiments, the ligand-binding protein comprises a mutation that decreases bicarbonate binding compared to a naturally occurring counterpart. In non-limiting examples, the modified cmBicarbBP6 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: I17X, W50X, Q72X, and T141X, where X is any amino acid, an amino acid that results in a conservative substitution, or an alanine, and where each position is counted in cmBicarbBP6 with the signal peptide replaced with a methionine (SEQ ID NO: 20 or 80). In some embodiments, the modified cmBicarbBP6 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the following substitutions: I17A, I17C, I17M, I17F, I17Y, I17E, I17W, W50A, W50C, W50F, W50Y, Q72C, T143A, T143C, T143F, T143Y, T143W, T143Q, T143E, and T143V. 
     mhFeBP1 is a non-limiting reference protein respect to bicarbonate-binding proteins. An alignment of mhFeBP1 with other polypeptides is provided in  FIG. 6A . 
     In various embodiments, a ligand-binding protein (or its naturally occurring counterpart) comprises
         (a) an amino acid sequence that is preferably (i) at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, and (ii) less than about 75%, 70%, 65%, 60%, 55%, 50%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, or 35% identical to mhFeBP1;   (b) a cysteine substitution (compared to its naturally occurring counterpart) within a stretch of at least 5, 10, or 20 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 9 of mhFeBP1;   (c) a stretch of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 9 of mhFeBP1;   (d) a stretch of amino acids in the sequence VYSXR (where X is any amino acid, or where X is Y, S, A or G) (SEQ ID NO: 160);   (e) a cysteine substitution (compared to its naturally occurring counterpart) within a stretch of at least 5, 10, or 20 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 100 of mhFeBP1;   (f) a stretch of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 100 of mhFeBP1;   (g) a stretch of amino acids in the sequence GLX 1 X 2 R (where X 1  is any amino acid, or where X 1  is T or S; and where X 2  is any amino acid, or where X 2  is T, K, R or G) (SEQ ID NO: 161);   (h) a cysteine substitution (compared to its naturally occurring counterpart) within a stretch of at least 5, 10, or 20 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 200 of mhFeBP1;   (i) a stretch of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 200 of mhFeBP1;   (j) a stretch of amino acids in the sequence YYX 1 X 2  (where X 1  is any amino acid, or where X 1  is Y, M, L, or I; and where X 2  is any amino acid, or where X 2  is G, A or Y) (SEQ ID NO: 162);   (k) no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15 deleted or inserted amino acids compared to mhFeBP1, not including amino acids added to the N-terminus or C-terminus of the polypeptide compared to its natural counterpart, and including or not including the signal peptide of the natural counterpart of the polypeptide;   (l) at least 7, 8, 9, or 10, or exactly 7, 8, 9, or 10 α-helices; and/or   (m) at least 4, 5, 6, or 7 β-strands or exactly 4, 5, 6, or 7 β-strands.       

     In embodiments, two or more or each of features (b)-(j) above occurs in the polypeptide in the order listed above as the amino acid sequence of the polypeptide is viewed or read from the N-terminus to the C-terminus (with additional features and/or amino acid sequences therebetween). For example, the polypeptide may have an N-terminus, followed by feature (b), (c), or (d), followed by feature (e), (f), or (g), followed by feature (h), (i), or (j), followed by the C-terminus. 
     The features listed above are not limiting and may be combined with any other relevant features disclosed herein, including those listed below. 
     In some embodiments the polypeptide comprises the following sequence: 
                    XXmXXnVYSXEXXXlikpXlXXFXXXtGIX!#XXXXXXXeLv#RlXXEGX               XtpADvflTXDagXlXXaX#aXllqpX#srel#XX!PXqfRXX#nXWfGL               sXRXr!XvynkXrVkpXqXXXXyXdLtXpqwkXk!avrXsXnXYnqslXX               XXXaXXGX#XtkqflXGLkaNXarXpXgXdXXqvXAvaXGXXXXXIXNhY               YXXrXlXXXXeX#XXXaXaaXXXfpXXXXXGahXN!sGigvtXasknXeX               AXXliE%$vXXXaQXm%AXl#XEYPvXXX!XXXpXlXXXgtFrXXX!Xla               XlaenXeaalXlX#XvgXrXX            
wherein each
         X is, individually, any amino acid or is absent,   ! is. individually, I or V,   $ is, individually, L or M,   % is, individually, F or Y. and   # is, individually, N, D, Q, or E.       

     In a non-limiting example, the ligand-binding protein comprises an N-terminal domain and a C-terminal domain connected by a flexible hinge, with the ligand-binding site (the ligand binding domain) located in the cleft between the N-terminal and the C-terminal domain. 
     In some embodiments, the ligand-binding protein comprises, from the N-terminus to the C-terminus, a first β-strand (β1), followed by a first α-helix (α1), followed by a second β-strand (β2), followed by a second α-helix (α2), followed by a third β-strand (β3), followed by a third α-helix (α3), followed by a fourth α-helix (α4), followed by a fourth β-strand (β4), followed by a fifth α-helix (α5), followed by a sixth α-helix (α6), followed by a fifth β-strand (β5), followed by a seventh α-helix (α7), followed by a sixth β-strand (β6), followed by a seventh β-strand (β7), followed by an eighth α-helix (α5), followed by a ninth α-helix (α9), followed by a tenth α-helix (α10). In some embodiments, the polypeptide comprises (i) 1, 2, or 3 amino acid substitutions between β1 and α1; (ii) 1, 2, or 3 amino acid substitutions between α1 and β2; (iii) 1, 2, or 3 amino acid substitutions between β2 and α2; (iv) 1, 2, or 3 amino acid substitutions between α2 and β3; (v) 1, 2, or 3 amino acid substitutions between the β3 and α3: (vi) 1, 2, or 3 amino acid substitutions between α3 and α4; (vii) 1, 2, or 3 amino acid substitutions between α4 and β4; (viii) 1, 2, or 3 amino acid substitutions between β4 and α5; (ix) 1, 2, or 3 amino acid substitutions between α5 and α6; (x) 1, 2, or 3 amino acid substitutions between α6 and β5; (xi) 1, 2, or 3 amino acid substitutions between β5 and α7; (xii) 1, 2, or 3 amino acid substitutions between α7 and β6; (xiii) 1, 2, or 3 amino acid substitutions between β6 and β7; (xiv) 1, 2, or 3 amino acid substitutions between β7 and α8; (xv) 1, 2, or 3 amino acid substitutions between α8 and α9; (xvi) 1, 2, or 3 amino acid substitutions between α9 and α10; (xvii) 1, 2, or 3, amino acid substitutions in α1, α2, α3, α4, α5, α6, α7, α8, α9, or α10; and/or (xviii) 1, 2, or 3 amino acid substitutions in β1, β2, β3, β4, β5, β6, or β7. In some embodiments, the substitutions are conservative substitutions. In various embodiments, the polypeptide comprises a cysteine substitution within β1, between β1 and α1, within α1, between β2 and α2, between β2 and α3, within β4, within α5, within α6, within β5, within α7, or between α8 and α9. 
     The ligand-binding polypeptide may further comprise 1, 2, or more iron binding sites. In some embodiments, said iron comprises Fe(I), Fe(II), or Fe(III). 
     In embodiments, a biosensor comprises a modified mhFeBP1. In non-limiting examples, the modified mhFeBP1 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: Y10X, Q12X, Y14X, L15X, A36X, D37X, V59X, C135X, R137X, N141X, S142X, G178X, C191X, N197X, N267X, and E269X, where X is any amino acid. an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in mhFeBP1 with the signal peptide replaced with a methionine (SEQ ID NO: 21 or 81). The sequence for mhFeBP1 (SEQ ID NO: 21 or 81) comprises C135A and C191A mutations. In some embodiments, the modified mhFeBP1 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 of the following substitutions: Y10C, Y10A, Y10M, Y10F, Y10I, Y10E, Y10W, Q12C, Y14C, L15C, A36C, D37A, D37F, D37Y, D37C, V59C, C135A, R137C, N141C, N141A, N141F, N141Y, N141W, N141Q, N141E, N141V, S142C, G178C, C191A, N197C, N267C, and E269C. 
     In embodiments, a biosensor comprises a modified exiFeBP2. In non-limiting examples, the modified exiFeBP2 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: S9X, H11X, D13X, V14X, G35X, K36X, A59X, R136X, N140X, M141X, N177X, N196X, N269X, and E271X, where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in exiFeBP2 with the signal peptide replaced with a methionine (SEQ ID NO: 22 or 82). In some embodiments, the modified exiFeBP2 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of the following substitutions: S9C, S9A, S9M, S9F, S91, S9E, S9W, H11C, D13C, V14C, G35C, K36C, K36A, K36F, K36Y, A59C, R136C, N140C, N140A, N140F, N140Y, N140W, N140Q, N140E, N140V, M141C, N177C, N196C, N269C, and E271C. 
     In embodiments, a biosensor comprises a modified teFeBP3. In non-limiting examples, the modified teFeBP3 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: A8X, H10X, D12X, T13X, E35X, A36X, V58X, R135X, N139X, I140X, N176X, C184X, N195X, N268X, and E270X, where X is any amino acid, an amino acid that results in a conservative substitution. or a cysteine, and where each position is counted in teFeBP3 with the signal peptide replaced with a methionine (SEQ ID NO: 23 or 83). The sequence for teFeBP3 (SEQ ID NO: 23 or 83) comprises a C184S mutation. In some embodiments, the modified teFeBP3 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the following substitutions: A8C, A8S, A8M, A8F, A8I, A8E. A8W, H10C, D12C, T13C, E35C, A36C, A36K, A36F. A36Y, V58C, R135C, N139C, N139A, N139F, N139Y, N139W, N139Q, N139E, N139V, I140C, N176C, C184A, C184S, N195C, N268C, and E270C. 
     In embodiments, a biosensor comprises a modified cnFeBP4. In non-limiting examples, the modified cnFeBP4 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: G8X, A10X, R12X, L13X, S34X, G35X, N58X, P135X, S139X, E140X, K176X, N195X, D264X, and E266X, where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in cnFeBP4 with the signal peptide replaced with a methionine (SEQ ID NO: 24 or 84). In some embodiments, the modified cnFeBP4 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 of the following substitutions: G8C, G8A, G8M, G8F, G81, G8E, G8W, A10C, R12C, L13C, S34C, G35C, G35K, G35F, G35Y, N58C, P135C, S139C, S139A, S139F, S139Y, S139W, S139Q, S139E, S139V, E140C, K176C, N195C, D264C, and E266C. 
     ttFeBP5 is a non-limiting reference protein respect to bicarbonate-binding proteins. An alignment of ttFeBP5 with other polypeptides is provided in  FIG. 6B . 
     In various embodiments, a ligand-binding protein (or its naturally occurring counterpart) comprises
         (a) an amino acid sequence that is preferably (i) at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, and (ii) less than about 75%, 70%, 65%, 60%, 55%, 50%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, or 35% identical to ttFeBP5;   (b) a cysteine substitution (compared to its naturally occurring counterpart) within a stretch of at least 5, 10, or 20 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 9 of ttFeBP5;   (c) a stretch of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 9 of ttFeBP5;   (d) a stretch of amino acids in the sequence YXGR (where X is any amino acid, or where X is S or T) (SEQ ID NO: 163);   (e) a cysteine substitution (compared to its naturally occurring counterpart) within a stretch of at least 5, 10, or 20 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 53 of ttFeBP5;   (f) a stretch of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 53 of ttFeBP5;   (g) a stretch of amino acids in the sequence SPAD (SEQ ID NO: 164);   (h) a cysteine substitution (compared to its naturally occurring counterpart) within a stretch of at least 5, 10, or 20 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 135 of ttFeBP5;   (i) a stretch of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids having at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, or 95% identity to a stretch of consecutive amino acids including position 135 of ttFeBP5;   (j) a stretch of amino acids in the sequence GWX 1 PX 2 Y (where X 1  is any amino acid, or where X 1  is T or A; and where X 2  is any amino acid, or where X 2  is T, A, or S) (SEQ ID NO: 165);   (k) no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15 deleted or inserted amino acids compared to ttFeBP5, not including amino acids added to the N-terminus or C-terminus of the polypeptide compared to its natural counterpart, and including or not including the signal peptide of the natural counterpart of the polypeptide;   (l) at least 9, 10, 11, 12, or 13, or exactly 9, 10, 11, 12, or 13 α-helices; and/or   (m) at least 5, 6, 7, 8, or 9, 3-strands or exactly 5, 6, 7, 8, or β-strands.       

     In embodiments, two or more or each of features (b)-(j) above occurs in the polypeptide in the order listed above as the amino acid sequence of the polypeptide is viewed or read from the N-terminus to the C-terminus (with additional features and/or amino acid sequences therebetween). For example, the polypeptide may have an N-terminus, followed by feature (b), (c), or (d), followed by feature (e), (f), or (g), followed by feature (h), (i), or (j), followed by the C-terminus. 
     The features listed above are not limiting and may be combined with any other relevant features disclosed herein, including those listed below. 
     In some embodiments the polypeptide comprises the following sequence: 
                    XXXMLTXYSGRGXXLVXXLVXQXEX#XXDXXVXVRYXXDX#LLAALQ#EG               DXSPADVFWANTAGALGXAXXXGLLXXLXXXLTXXXXRFXPXXXXWXPVS               XRXRVXAYNXXXXSDX#LPDSX$DLPEXXEEXGXXXRXGWTPXYSSFQDF               !TA$RXXEGEEATXAWLXXMXAAGXXSYPS#XAMX#AIXAGE!DXAXTNH               YY!QRXLXGXXXXXXXXXXXXXXXXXXXXXXXXXXXAXAXXGTXXFXXGD               AGXLALVTGAGXLXTSXXXTXAXRFLRXLLSXXAQX%FAXXXXEYPL!XG               VXXXXX$XPXD#AXXXSPXIDLXKLXD$#PTLDL$R#AGVXL            
wherein each
         X is, individually, any amino acid or is absent,   ! is, individually, I or V,   $ is, individually, L or M,   % is, individually, F or Y, and   # is, individually, N, D, Q, or E.       

     In a non-limiting example, the ligand-binding protein comprises an N-terminal domain and a C-terminal domain connected by a flexible hinge, with the ligand-binding site (the ligand binding domain) located in the cleft between the N-terminal and the C-terminal domain. 
     In some embodiments, the ligand-binding protein comprises, from the N-terminus to the C-terminus: a first β-strand (β1), followed by a first α-helix (α1), followed by a second β-strand (β2), followed by a second α-helix (2), followed by a third β-strand (β3), followed by a third α-helix (α3), followed by a fourth α-helix (α4), followed by a fourth β-strand (β4), followed by a fifth α-helix (α5), followed by a sixth α-helix (α6), followed by a fifth β-strand (β5), followed by a seventh α-helix (α7), followed by a eighth α-helix (α8), followed by a sixth β-strand (β6), followed by a ninth α-helix (α9), followed by a seventh β-strand (β7), followed by a tenth α-helix (α10), followed by an eleventh α-helix (α11), followed by an eighth β-strand (β8), followed by a ninth β-strand (β9), followed by a twelfth α-helix (α12), followed by a thirteenth α-helix (α13). In some embodiments, the polypeptide comprises (i) 1, 2, or 3 amino acid substitutions between β1 and α1; (ii) 1, 2, or 3 amino acid substitutions between α1 and β2; (iii) 1, 2, or 3 amino acid substitutions between β2 and α2; (iv) 1, 2, or 3 amino acid substitutions between α2 and β3; (v) 1, 2, or 3 amino acid substitutions between the β3 and α3; (vi) 1, 2, or 3 amino acid substitutions between α3 and α4; (vii) 1, 2, or 3 amino acid substitutions between α4 and β4: (viii) 1, 2, or 3 amino acid substitutions between β4 and α5; (ix) 1, 2, or 3 amino acid substitutions between α5 and α6; (x) 1, 2, or 3 amino acid substitutions between α6 and β5; (xi) 1, 2, or 3 amino acid substitutions between β5 and α7; (xii) 1, 2, or 3 amino acid substitutions between α7 and α8; (xiii) 1, 2, or 3 amino acid substitutions between α8 and β6; (xiv) 1, 2, or 3 amino acid substitutions between β6 and α9; (xv) 1, 2, or 3 amino acid substitutions between α9 and β7; (xvi) 1, 2, or 3 amino acid substitutions between β7 and α10; (xvii) 1, 2, or 3 amino acid substitutions between α10 and α11; (xviii) 1, 2, or 3 amino acid substitutions between α11 and β8; (xix) 1, 2, or 3 amino acid substitutions between β8 and β9; (xx) 1, 2, or 3 amino acid substitutions between β9 and α12; (xxi) 1, 2, or 3 amino acid substitutions between α12 and α13; (xxii) 1, 2, or 3, amino acid substitutions in α1, α2, α3, α4, α5, α6, α7, α8, α9, α10, α11, α12, or α13; and/or (xxiii) 1, 2, or 3 amino acid substitutions in β1, β2, β3, β4, β5, β6, β7, β8, or β9. In some embodiments, the substitutions are conservative substitutions. In various embodiments, the polypeptide comprises a cysteine substitution within β1, between β1 and α1, within α1, within β2, within β4, within β5, between β4 and α7, within α7, within β6, within α9, or between α11 and β8. 
     The ligand-binding polypeptide may further comprise 1, 2, or more iron binding sites. In some embodiments, said iron comprises Fe(I), Fe(II), or Fe(III). 
     In embodiments, a biosensor comprises a modified ttFeBP5. In non-limiting examples, the modified ttFeBP5 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: G10X, G12X, S14X, L15X, S36X, T37X, N60X, V137X, P141X, T142X, S179X, N198X, 1263X, and E265X, where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in ttFeBP5 with the signal peptide replaced with a methionine (SEQ ID NO: 25 or 85). In some embodiments, the modified ttFeBP5 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the following substitutions: G10C, G10A, G10M, G10F, G10I, G10E, G10W, G12C, S14C, L15C, S36C, T37C, T37K, T37F, T37Y, N60C, V137C, P141C, P141A, P141F, P141Y, P141W, P141Q. P141E, P141V, T142C, S179C, N198C, 1263C, and E265C. 
     In embodiments, a biosensor comprises a modified msFeBP6. In non-limiting examples, the modified msFeBP6 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: G8X, S10X, A12X, L13X, G34X, R35X, N58X, I132X, P136X, T137X, S174X, N193X, V283X, and E285X, where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in msFeBP6 with the signal peptide replaced with a methionine (SEQ ID NO: 26 or 86). In some embodiments, the modified msFeBP6 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the following substitutions: G8C, G8A, G8M, G8F, G81, G8E, G8W, S10C, A12C, L13C, G34C, R35C, R35K, R35F, R35Y, N58C, I132C, P136C, P136A, P136F, P136Y, P136W, P136Q, P136E, P136V, T137C, S174C, N193C, V283C, and E285C. 
     In embodiments, a biosensor comprises a modified srFeBP7. In non-limiting examples, the modified srFeBP7 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: G7X, S9X, A11X, L12X, G33X, T34X, N57X, V131X, P135X, A136X, S174X, N192X, V285X, and E287X, where X is any amino acid, an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in srFeBP7 with the signal peptide replaced with a methionine (SEQ ID NO: 27 or 87). In some embodiments, the modified srFeBP7 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the following substitutions: G7C, G7A, G7M, G7F, G71, G7E, G7W, S9C, A11C, L11C, G33C, T34C, T34K, T34F. T34Y, N57C, V131C, P135C, P135A, P135F, P135Y, P135W, P135Q, P135E, P135V, A136C, S174C, N192C, V285C, and E287C. 
     In embodiments, a biosensor comprises a modified hlFeBP8. In non-limiting examples, the modified hlFeBP8 may comprise one or more, or any combination of the following substitutions compared to its naturally occurring counterpart: G7X, G9X, F11X, L12X, A34X, G35X, V58X, L129X, P133X, S134X, C138X, D171 X, C176C, N190X, T256X, and E258X, where X is any amino acid. an amino acid that results in a conservative substitution, or a cysteine, and where each position is counted in hlFeBP8 with the signal peptide replaced with a methionine (SEQ ID NO: 28 or 88). The sequence for hlFeBP8 (SEQ ID NO: 28 or 88) comprises C138A and C176A mutations. In some embodiments, the modified hlFeBP8 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the following substitutions: G7C, G7A, G7M, G7F, G71, G7E, G7W, G9C, F11C, L12C, A34X. G35C. G35K, G35F, G35Y, V58C, L129C, P133C, P133A, P133F, P133Y, P133W, P133Q, P133E, P133V, S134C, C138A, D171C, C176A, N190C, T256C, and E258C. 
     Reporter Group Attachment 
     Aspects of the present subject matter provide a biosensor that comprises a one or more reporter groups attached to a ligand-binding protein, wherein binding of a ligand to a ligand-binding domain of the ligand-binding protein causes a change in signaling by the reporter group. In various embodiments, the reporter group is attached to an endosteric site. an allosteric site, or a peristeric site of the ligand-binding protein. In embodiments, the reporter group is covalently or noncovalently attached to the ligand-binding protein. 
     As used herein, “signaling” refers to the emission of energy (which may be referred to as a “signal”) by one or more reporter groups. In various implementations, the signal comprises electromagnetic radiation such as a light. In some embodiments, the signal is detected as a complete emission spectrum (or spectrums) or a portion (or portions) thereof. 
     For example, a signal may comprise emitted light at a particular wavelength or wavelengths, or range(s) of wavelengths. In some embodiments, a change in signaling comprises a spectral change (e.g., a spectral shift and/or change in intensity). In some embodiments, a change in signaling comprises a dichromatic shift or a monochromatic fluorescence intensity change. 
     For convenience and depending on context, a reporter group may be referred to by a name of an unattached form of the reporter group regardless of whether the reporter group is attached to a ligand-binding protein. For example, a compound known as “Compound A” when in an unconjugated form may be referred to herein as “Compound A” when in a form that is attached to a ligand-binding protein. In a specific example, the term “Acrylodan” is used to refer to unreacted/unconjugated Acrylodan, as well as Acrylodan that is conjugated to a ligand-binding protein. 
     In certain embodiments, a biosensor comprises a reporter group that is conjugated to a ligand-binding protein, and the reporter group is conjugated to an amino acid of the protein that is at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 angstroms (Å) from the ligand when the ligand is bound to the protein. In embodiments, the reporter group is conjugated to an amino acid of the protein that is about 0.1 Å to about 100 Å, about 0.1 Å to about 5 Å, about 5 Å to about 10 Å, about 10 Å to about 20 Å. about 20 Å to about 50 Å, about 50 Å to about 75 Å, or about 75 Å to about 100 Å from the ligand when the ligand is bound to the protein. In some embodiments, the reporter group is conjugated to an amino acid of the protein that is within an α-helix or a β-strand. In some embodiments, the reporter group is conjugated to an amino acid that (i) is not within an α-helix or a β-strand, but is within about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids of an amino acid of the protein&#39;s amino acid sequence that is within an α-helix or a β-strand. In some embodiments, the reporter group is conjugated to an amino acid that is in an inter-domain hinge amino acid region between two domains of a protein. In some embodiments, the reporter group is conjugated to an amino acid that is between (i) an α-helix and a β-strand; (ii) two α-helixes, or (iii) two β-strands of a protein. In some embodiments, the reporter group is conjugated to an amino acid (e.g., a cysteine such as a cysteine added by substitution compared to a naturally corresponding polypeptide) between positions 1-25, 25-50, 50-75, 75-100, 100-125, 125-150, 150-175, 175-200, 200-225, 225-250, 250-275, 275-350, 275-300, 275-325, 300-325, 300-350, 300-400, 350-400, 350-400, or 400-450 (inclusive) of a polypeptide (e.g., not including N-terminal fusion proteins compared to the polypeptide&#39;s naturally occurring counterpart). 
     Periplasmic binding proteins are characterized by two lobes connected by a hinge region: ligand bind at a location at the interface between the two domains. Such proteins or engineered versions thereof (as described herein) can adopt two different conformations: a ligand-free open form and a ligand-bound closed form, which interconvert through a relatively large bending motion around the hinge (FIG. 1A; Dwyer et al., 2004. Current Opinion in Structural Biology 12:495-504). 
     The remarkable adaptability of this superfamily of ligand-binding proteins is likely to have arisen from positioning the location of binding of the ligand at the interface between the lobes and from the large ligand-mediated conformational change. In this arrangement, ligands are placed within an environment that resembles a protein interior, but the residues forming the contact points or contact sites with the ligand are positioned at the surface of the lobes. 
     Direct signaling relationships between proteins and reporter groups are readily designed by replacing a residue known to form a ligand contact with a cysteine to which the fluorophore is attached (“endosteric” attachment site). Other, indirect signaling relationships can be established in two ways. The first relies on visual inspection of the ligand complex structure, and identifying residues that are located in the vicinity of the binding site, but do not interact directly with the ligand, and that are likely to be involved in conformational changes. Typically, such “peristeric” sites are located adjacent to the residues that form direct contacts with the bound ligand. In the case of the bPBPs, such residues are located at the perimeter of the inter-domain cleft that forms the ligand binding site location. The environment of these peristeric sites changes significantly upon formation of the closed state. These are examples of positions which are proximal to the ligand-binding pocket/domain. The second, most general, approach identifies sites in the protein structure that are located anywhere in the protein, including locations at some distance away from the ligand-binding site (i.e., distal to the ligand-binding pocket/domain), and undergo a local conformational change in concert with ligand binding. If the structures of both the open and closed states are known, then such “allosteric” sites can be identified using a computational method that analyzes the conformational changes that accompany ligand binding (Marvin et al., Proc. Natl. Acad. Sci. USA 94:4366-4371, 1997). Alternatively, once allosteric sites have been identified in one bPBP, modeling and structural homology arguments can be invoked to identify such sites in other bPBPs in which only one state has been characterized (Marvin &amp; Hellinga. J. Am. Chem. Soc. 120:7-11, 1998). This generalized conformational analysis also may identify peristeric and endosteric sites, which were identified and classified by visual inspection. 
     In non-limiting implementations, the reporter group is attached to the ligand-binding protein via a biotin-avidin interaction. The reporter group may be, e.g. conjugated to biotin and the ligand-binding protein is conjugated to avidin. In an example, the avidin is bound to four biotin molecules wherein each biotin molecule is individually conjugated to a reporter group. Alternatively, the reporter group is conjugated to avidin and the ligand-binding protein is conjugated to biotin. For example, the avidin is bound to four biotin molecules, wherein each biotin molecule is individually conjugated to a ligand-binding protein. 
     As used herein, “conjugated” means covalently attached. One compound may be directly conjugated to another compound, or indirectly conjugated, e.g., via a linker. 
     In some embodiments, the reporter group is directly attached to the ligand-binding protein. In various embodiments, the reporter group is attached to an amino acid of the ligand-binding protein that is at least about 2, 4, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 angstroms (Å) from the ligand when the ligand is bound to the ligand-binding protein. In certain embodiments, the reporter group is conjugated to an amino acid having a position within positions 1-25, 25-50, 50-75, 75-100, 100-125, 125-150, 150-175, 175-200, 200-225, 225-250, 250-275, or 275-300 of the ligand-binding protein, wherein position 1 is the N-terminal amino acid of the ligand-binding protein. In non-limiting examples, the reporter group is conjugated to an amino acid of the ligand-binding protein that is (a) within an α-helix or a β-strand of the ligand-binding protein; (b) not within an α-helix: (c) not within a β-strand; (d) within about 5 or 10 amino acids of an amino acid that is within an α-helix or β-strand; (e) within a stretch of consecutive amino acids that links two domains of the ligand-binding protein; (f) within a stretch of consecutive amino acids that links an α-helix and a β-strand; (g) within a stretch of consecutive amino acids that links two α-helices: or (h) within a stretch of consecutive amino acids that links two β-strands. In some embodiments, the reporter group is directly attached to the N-terminus or the C-terminus of the ligand-binding protein. 
     The reporter group may be conjugated to the ligand-binding protein a variety of linkers or bonds, including (but not limited to) a disulfide bond, an ester bond, a thioester bond, an amide bond, or a bond that has been formed by a click reaction. In some embodiments, the click reaction is a reaction between (a) an azide and an alkyne; (b) an azide and an alkyne in the presence of Cu(I); (c) an azide and a strained cyclooctyne; (d) an azide and a dibenzylcyclooctyne, a difluorooctyne, or a biarylazacyclooctynone; (e) a diaryl-strained-cyclooctyne and a 1,3-nitrone: (f) an azide, a tetrazine, or a tetrazole and a strained alkene; (g) an azide, a tetrazine, or a tretrazole and a oxanorbornadiene, a cyclooctene, or a trans-cycloalkene: (h) a tetrazole and an alkene; or (i) a tetrazole with an amino or styryl group that is activated by ultraviolet light and an alkene. These exemplary click chemistry reactions have high specificity, efficient kinetics, and occur in vivo under physiological conditions. See, e.g., Baskin et al. Proc. Natl. Acad. Sci. USA 104(2007):16793; Oneto et al. Acta biomaterilia (2014); Neves et al. Bioconjugate chemistry 24(2013):934; Koo et al. Angewandte Chemie 51(2012): 11836; Rossin et al. Angewandte Chemie 49(2010):3375, and U.S. Patent Application Publication No. 20160220686, published Aug. 4, 2016, the entire content of each of which is incorporated herein by reference. For a review of a wide variety of click chemistry reactions and their methodologies, see e.g., Nwe K and Brechbiel M W, 2009 Cancer Biotherapy and Radiopharmaceuticals, 24(3): 289-302: Kolb H C et al., 2001 Angew. Chem. Int. Ed. 40: 2004-2021. The entire contents of each of the foregoing references are incorporated herein by reference. 
     As used herein, the term “linker” refers to a molecule or sequence (such as an amino acid sequence), that attaches, as in a bridge, one molecule or sequence to another molecule or sequence. “Linked” means attached or bound by covalent bonds, or non-covalent bonds, or other bonds, such as van der Waals forces. In some embodiments, a linker comprises a chemical structure that has resulted from a reaction used to attach one molecule to another. 
     In various implementations of the present subject matter, the reporter group is conjugated to a cysteine of the ligand-binding protein. The cysteine may be present in the amino acid sequence of a natural counterpart or version of the ligand-binding protein or added to the ligand-binding protein by a substitution mutation in a coding sequence or by altering the sequence synthetically using known chemical means. In some embodiments, the cysteine is at the N-terminus or the C-terminus of the ligand-binding protein. In some embodiments, the cysteine is no more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids from the N-terminus or the C-terminus of the ligand-binding protein, In some embodiments, the cysteine is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids from the N-terminus or the C-terminus of the ligand-binding protein. 
     Non-limiting examples relate to the conjugation of a reporter group to a primary amine of the ligand-binding protein. In certain embodiments, the primary amine is present in a lysine of the ligand-binding protein. The lysine may be present in the amino acid sequence of a natural counterpart or version of the ligand-binding protein or added to the ligand-binding protein by a substitution mutation in a coding sequence or by altering the sequence synthetically using known chemical means. In some embodiments, the lysine is at the N-terminus or the C-terminus of the ligand-binding protein. In some embodiments, the lysine is no more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids from the N-terminus or the C-terminus of the ligand-binding protein. In some embodiments, the lysine is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 5-15, 5-20, 5-25, 5-100, 10-15, 10-20, 10-25, 10-50, 10-100, 25-50, 25-75, or 25-100 amino acids from the N-terminus or the C-terminus of the ligand-binding protein. 
     Aspects of the present subject matter provide a biosensor in which the reporter group is attached to the ligand-binding protein via a linker. In some embodiments, the linker comprises an organic compound that is less than about 30, 20, 15, or 10 Å long. Non-limiting examples of linkers include O, S, NH, PH, and alkyl linkers. 
     “Alkyl,” as used herein, refers to the radical of saturated or unsaturated aliphatic groups, including straight-chain alkyl, alkenyl, or alkynyl groups, branched-chain alkyl, alkenyl, or alkynyl groups. cycloalkyl, cycloalkenyl, or cycloalkynyl (alicyclic) groups, alkyl substituted cycloalkyl, cycloalkenyl, or cycloalkynyl groups, and cycloalkyl substituted alkyl, alkenyl, or alkynyl groups. Unless otherwise indicated, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C 1 -C 30  for straight chain, C 3 -C 30  for branched chain), more preferably 20 or fewer carbon atoms, more preferably 12 or fewer carbon atoms, and most preferably 8 or fewer carbon atoms. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure. The ranges provided above are inclusive of all values between the minimum value and the maximum value. The term “alkyl” includes both “unsubstituted alkyls” and “substituted alkyls,” the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino, amido. amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. Unless the number of carbons is otherwise specified. “lower alkyl” as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Preferred alkyl groups are lower alkyls. The alkyl groups may also contain one or more heteroatoms within the carbon backbone. Preferably the heteroatoms incorporated into the carbon backbone are oxygen, nitrogen, sulfur, and combinations thereof. In certain embodiments, the alkyl group contains between one and four heteroatoms. 
     In some embodiments, the linker comprises a bond formed by a chemical reaction involving a reactive group such as a maleimide group. Alternatively or in addition, the linker comprises a stretch of amino acids. In a non-limiting example, the linker comprises a polyglycine linker. In embodiments, the polyglycine linker comprises 2, 3, 4, 5, or more glycines. Optionally, the polyglycine linker further comprises a serine. 
     In various implementations, the reporter group is attached to a linker via a covalent bond and the linker is attached to a ligand-binding protein via a covalent bond. In embodiments, the covalent bond between the linker and the reporter group and/or the covalent bond between the linker and the ligand-binding protein is a disulfide bond, an ester bond, a thioester bond, an amide bond, a carbamate bond, or a bond that has been formed by a click reaction. Non-limiting examples of click reactions include reactions between an azide and an alkyne; an azide and an alkyne in the presence of Cu(I); an azide and a strained cyclooctyne; an azide and a dibenzylcyclooctyne, a difluorooctyne, or a biarylazacyclooctynone; a diaryl-strained-cyclooctyne and a 1,3-nitrone; an azide, a tetrazine, or a tetrazole and a strained alkene; an azide, a tetrazine, or a tretrazole and a oxanorbornadiene, a cyclooctene, or a trans-cycloalkene: a tetrazole and an alkene: or a tetrazole with an amino or styryl group that is activated by ultraviolet light and an alkene. 
     Reporter Groups 
     Various types of reporter groups may be used in embodiments of the present subject matter. For example, the reporter group may comprise a fluorophore that produces a fluorescent signal. Biosensors comprising a fluorophore may be referred to herein as fluorescently responsive sensors (FRSs). 
     Preferably, the binding of ligand to an FRS results in a change in ratiometric ΔR in the signal from a reporter group. A ratiometric signal (R 1,2 ) is defined as the quotient of two intensities, I λ1  and I λ2 , measured at two independent wavelengths. λ 1  and λ 2  and may be calculated according to the following equation: 
     
       
      
       R 
       1,2 
       =I 
       λ1 
       /I 
       λ2  
      
     
     In some embodiments, intensities are, e.g., integrated, filtered, assessed, detected, or evaluated over a range of wavelengths. In some embodiments, intensities are integrated over a range of wavelengths in a recorded emission spectrum. In some embodiments, a range of wavelengths is selected using a filter. In some embodiments, λ 1  is the intensity over a 1 nm to 60 nm interval centered between 400 and 1000 nm, and λ 2  is the intensity over a 1 nm to 60 nm interval centered between 400 nm and 1000 nm. In some embodiments, intensities are integrated, filtered, assessed, detected, or evaluated over a 1 nm, 2 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 75 nm, 100 nm, 10-40 nm, 10-50 nm, 20-50 nm, or 10-100 nm regions, centered between 400-1000 nm, e.g. between 420 nm and 520 nm for λ 1 , and 400-1000 nm, e.g. between 500 nm to 600 nm for λ 2 . In some embodiments, intensities are recorded through a bandpass filter. A non-limiting example of a bandpass filter is a 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 75 nm, 100 nm, 10-40 nm, 10-50 nm, 20-50 nm, or 10-100 nm bandpass filter, centered between 400-1000 nm, e.g. at 452 nm for λ 1  and at 400-1000 nm, e.g. at 528 nm (2). 
     Aspects of the present subject matter provide FRSs whose emission spectra change (e.g., the shape of the emission spectra change) in response to ligand binding. In various embodiments, the ratio of intensities at two chosen wavelengths of an FRS&#39;s emission spectrum changes upon ligand binding. In some embodiments, the emission spectral shape and/or intensity of the fluorophore changes when the position of atoms within the fluorophore changes with respect to each other (e.g., due to the rotation of bound atoms with respect to each other or a change in the angle of a bond). In non-limiting examples, the emission spectral shape and/or intensity of the fluorophore changes when (i) one portion of the fluorophore rotates around a bond axis compared to another portion of the fluorophore and/or (ii) when the angle of a bond between two atoms of the fluorophore changes. In a non-limiting example, the fluorophore is a prodan-derived fluorophore (e.g., Acrylodan or Badan) and binding of ligand alters the orientation of a dimethylamino group, a naphthalene ring, and/or a carbonyl with respect to the ligand-binding protein and/or each other. In a non-limiting example, the degree of polarization of a dipole on the fluorophore changes in response to ligand binding. In various embodiments, the emission spectral shape and/or intensity of the fluorophore changes when an atom electrostatically interacts with the fluorophore. For example, the emission spectral shape and/or intensity of the fluorophore changes when the source of a positive or negative charge changes its distance with respect to the fluorophore within about 1, 2, 3, 4, 5, or 10 Å of the fluorophore. In some embodiments, the fluorophore exhibits hypsochromicity or bathochromicity upon ligand binding to the ligand-binding domain of the ligand-binding protein. In certain embodiments, the fluorophore has an emission spectrum comprising radiation with a wavelength (e.g., a peak emission wavelength) of about 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, 510 nm 520 nm, 530 nm, 540 nm, 550 nm, 560 nm, 570 nm, 580 nm, 590 nm, 600 nm, 610 nm, 620 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670 nm, 680 nm, 690 nm, 700 nm, 710 nm, 720 nm, 730 nm, 740 nm, 750 nm, 760 nm, 770 nm, 780 nm, 790 nm, 800 nm, 850 nm, 900 nm, 950 nm, or 1000 nm, or about 400 nm to about 450 nm, about 450 nm to about 500 nm, about 500 nm to about 550 nm, about 550 nm to about 60) nm, about 600 nm to about 650 nm, about 650 to about 700 nm, about 700 nm to about 750 nm, about 750 nm to about 800 nm, or about 800 nm to about 1000 nm. 
     In some embodiments, the signal comprises the emission intensity of the fluorophore recorded at a single wavelength or range of wavelengths. The change in signal may be a shift in the single wavelength or range of wavelengths. In some embodiments, the shift in the wavelength is at least about 1 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 11 nm, at least about 12 nm, at least about 13 nm, at least about 14 nm, at least about 15 nm, at least about 16 nm, at least about 17 nm, at least about 18 nm, at least about 19 nm, at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, at least about 100 nm, at least about 105 nm, at least about 110 nm, at least about 115 nm, at least about 120 nm, at least about 125 nm, or at least about 130 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 20 nm, about 2 nm to about 20 nm. about 3 nm to about 20 nm, about 4 nm to about 20 nm, about 5 nm to about 20 nm. about 1 nm to about 19 nm, about 1 nm to about 18 nm. about 1 nm to about 17 nm, 1 nm to about 16 nm, about 1 nm to about 15 nm, about 1 nm to about 14 nm, about 1 nm to about 13 nm, about 1 nm to about 12 nm, about 1 nm to about 11 nm, or about 1 nm to about 10 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 20 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 130 nm. 
     In certain embodiments, the signal comprises the ratio or quotient of the emission intensities recorded at two distinct wavelengths or ranges of wavelengths, i.e., a ratiometric signal. For example, as shown in  FIGS. 1A-D , ligand binding may be determined by measuring the ratio of blue to green emission intensities. The change in signal may be decreased emission intensity at one wavelength, and no change in emission intensity at the other wavelength. The change in signal may be increased emission intensity at one wavelength, and no change in emission intensity at the other wavelength. The change in signal may be increased emission intensity at one wavelength, and increased emission intensity at the other wavelength. The change in signal may be decreased emission intensity at one wavelength, and decreased emission intensity at the other wavelength. The change in signal may be increased emission intensity at one wavelength, and decreased emission intensity at the other wavelength. In some embodiments, the change in ratio of the emission intensities recorded at two distinct wavelengths or ranges of wavelengths may be at least about 1.1-fold, at least about 1.2-fold, at least about 1.4-fold, at least about 1.6-fold, at least about 1.8-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 12-fold, at least about 14-fold, at least about 16-fold, at least about 18-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 55-fold, at least about 60-fold, at least about 65-fold, at least about 70-fold, at least about 75-fold, at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 95-fold, or at least about 100-fold. In some embodiments, the change in ratio of the emission intensities recorded at two distinct wavelengths or ranges of wavelengths may be a decrease of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or of 5-25%, 25-50%, 25-75%, 50-75%, 50-90%, or 75-99% or the reciprocal thereof. 
     The change in signal may be a change in the ratio of the two distinct wavelengths or ranges of wavelengths. The change in signal may be a shift in the two distinct wavelengths or ranges of wavelengths. In some embodiments, one wavelength shifts. In some embodiments, both wavelengths shift. In some embodiments, the shift in the wavelength is at least about 1 nm, at least about 2 nm, at least about 3 nm, at least about 4 nm, at least about 5 nm, at least about 6 nm, at least about 7 nm, at least about 8 nm, at least about 9 nm, at least about 10 nm, at least about 11 nm, at least about 12 nm, at least about 13 nm, at least about 14 nm, at least about 15 nm, at least about 16 nm, at least about 17 nm, at least about 18 nm, at least about 19 nm, at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, at least about 100 nm, at least about 105 nm, at least about 110 nm, at least about 115 nm, at least about 120 nm, at least about 125 nm, or at least about 130 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 20 nm, about 2 nm to about 20 nm, about 3 nm to about 20 nm, about 4 nm to about 20 nm, about 5 nm to about 20 nm. about 1 nm to about 19 nm, about 1 nm to about 18 nm, about 1 nm to about 17 nm, 1 nm to about 16 nm, about 1 nm to about 15 nm, about 1 nm to about 14 nm, about 1 nm to about 13 nm, about 1 nm to about 12 nm. about 1 nm to about 11 nm, or about 1 nm to about 10 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 20 nm. In some embodiments, the shift in the wavelength is about 1 nm to about 130 nm. 
     A fluorophore may comprise, e.g., a fluorescent protein or an organic compound having a molecular weight less than about 2000 Daltons (Da). Non-limiting examples of commercially available fluorophores include such as 5-iodoacetamidofluorescein (5-IAF) or 6-iodoacetamidofluorescein (6-IAF), rhodamine, Oregon Green, eosin, Texas Red, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, Badan, Acrylodan, IAEDANS, comprising 3-cyano-7-hydroxycoumarin, 7-hydroxycoumarin-3-carboxylic acid, 6,8-difluoro-7-hydroxy-4-methylcoumarin, or 7-amino-4-methylcoumarin, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, DRAQ5, DRAQ7, or CyTRAK Orange, cascade blue, Nile red, Nile blue, cresyl violet, oxazine 170, proflavin, acridine orange, acridine yellow, auramine, crystal violet, malachite green, porphin, phthalocyanine, bilirubin, pyrene, N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-ox-a-1,3-diazol-4-yl)ethylenediamine (NBD), N-((2-(iodoacetoxy)ethyl)-N-methy-1)amino-7-nitrobenz-2-oxa-1,3-diazole (NBDE), JPW4039, JPW4042, JPW4045. Pacific Blue, CPM, N,N′-Dimethyl-N-(Iodoacetyl)-N′-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Ethylenediamine (IANBD), 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM). BODIPY 499, BODIPY 507/545, BODIPY 499/508, Alexa 432, Alexa488, Alexa532, Alexa546, Cy5, or 1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium methanesulfonate (PvMPO maleimide) (PyMPO). In various embodiments, the reporter group was thiol-reactive prior to being conjugated to a polypeptide disclosed herein. In embodiments, the reporter group is linked to a polypeptide disclosed herein via a disulfide bond. Additional non-limiting examples of commercially available fluorophores include fluorescent proteins such as Blue Fluorescent Protein (BFP), TagBFP, mTagBFP2, Azurite, Enhanced Blue Florescent Protein 2 (EBFP2), mKalama1, Sirius, Sapphire, T-Sapphire, Cyan Fluorescent Protein (CFP); Enhanced Cyan Fluorescent Protein (ECFP), Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFP1, AmCyan1. Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein (EGFP), Emerald, Superfolder GFP, AcGFP1, ZsGreen1, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, mNeonGreen. Yellow Fluorescent Protein (YFP), Enhanced Yellow Fluorescent Protein (EYFP), Citrine, Venus, Super Yellow Fluorescent Protein 2 (SYFP2), TagYFP, ZsYellow1, mBanana, Orange Fluorescent Protein (OFP), Monomeric Kusabira-Orange (mKO), mKOK, mKO2, mOrange, mOrange2, Red Fluorescent Protein (RFP), DsRed-Express, DsRed-Express2. DsRed2, AsRed2, mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato. TagRFP, TagRFP-T, mApple, mRuby, mRuby2, mPlum, HcRed-Tandem, mKate2, mNeptune, HcRed1, E2-Crimson, NirFP, TagRFP657, IFP1.4, or iRFP. 
     In some embodiments, the fluorophore comprises xanthene, a xanthene derivative. cyanine, a cyanine derivative, squaraine, a squaraine derivative, naphthalene, a naphthalene derivative, coumarin, a coumarin derivative, oxadiazole, an oxadiazole derivative, anthracene, an anthracene derivative, a boradiazaindacine (BODIPY) family fluorophore, pyrene, a pyrene derivative, acridine, an acridine derivative. arylmethine, an arylmethine derivative, tetrapyrrole, or a tetrapyrrole derivative. For example, the fluorophore may comprise a xanthene derivative comprising fluorescein or a fluorescein derivative, rhodamine. Oregon Green, eosin, or Texas Red. Non-limiting examples of fluorescein derivatives include 5-fluorescein, 6-carboxyfluorescein, 3′6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachlorofluorescein, 6-tetrachlorofluorescein, or isothiocyanate. In some embodiments, the fluorophore comprises a cyanine derivative comprising indocarbocyanine, oxacarbocyanine, thiacarbocyanine, or merocyanine. In certain embodiments, the fluorophore comprises a squaraine derivative comprising a ring-substituted squaraine. In various embodiments, the fluorophore comprises a naphthalene derivative comprising a dansyl or prodan naphthalene derivative. In a non-limiting example, the fluorophore comprises prodan or a derivative thereof. In certain embodiments, the fluorophore comprises Badan, Acrylodan, or N-(Iodoacetaminoethyl)-1-naphthylamine-5-sulfonic acid (IAEDANS). In some embodiments, the fluorophore comprises a coumarin derivative such as 3-cyano-7-hydroxycoumarin, 7-hydroxycoumarin-3-carboxylic acid, 6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU), or 7-amino-4-methylcoumarin. In various embodiments, the fluorophore comprises an oxadiazole derivative such as pyridyloxazole, nitrobenzoxadiazole, or benzoxadiazole. In certain embodiments, the fluorophore comprises an anthracene derivative comprising an anthraquinone such as DRAQ5, DRAQ7, or CyTRAK Orange. In various embodiments, the fluorophore comprises a pyrene derivative comprising cascade blue. In non-limiting examples the fluorophore comprises an oxazine derivative such as Nile red, Nile blue, cresyl violet, or oxazine 170. In some embodiments, the fluorophore comprises an acridine derivative such as proflavin, acridine orange, or acridine yellow. In certain embodiments, the fluorophore comprises an arylmethine derivative such as auramine, crystal violet, or malachite green. In various embodiments, the fluorophore comprises a tetrapyrrole derivative comprising porphin, phthalocyanine, or bilirubin. 
     Aspects of the present subject matter relate to the use of fluorophores that may readily be attached to a ligand-binding protein disclosed herein, e.g., at a cysteine residue. For example, a fluorophore may comprise a sulfhydryl group prior to attachment to a ligand-binding protein that is reacted with a moiety of the ligand-binding protein to attach the fluorophore to the ligand-binding protein. In some embodiments, the fluorophore comprised a thiol group prior to attachment to the ligand-binding protein. For example, the fluorophore was thiol reactive prior to attachment to the ligand-binding protein. Non-limiting examples of fluorophores that may readily be attached to ligand-binding proteins using thiol reactions include fluorescein, pyrene, NBD, NBDE, Acrylodan (6-acryloyl 1-2-dimethylaminonaphthalene), Badan (6-bromo-acetyl-2-dimethylamino-naphthalene), JPW4039, JPW4042, or JPW4045. 
     In certain embodiments, the fluorophore comprises a derivative of a Prodan-based fluorophore such as Acrylodan or Badan. The excitation and emission properties of the Prodan-based fluorophores Acrylodan and Badan can be altered by manipulating the fluorescent ring system, while preserving the dimethylamino donor group, and the twistable carbonyl acceptor (Klymchenko 2013  Progress in Molecular Biology and Translational Science,  35-58). Replacement of the two-ring naphthalene with a three-ring anthracene (Lu 2006  J. Org. Chem.,  71, 9651-9657), fluorene (Kucherak 2010.  J. Phys. Chem. Let.,  1, 616-620), pyrene (Niko 2013  Chem. Eur. J.,  19, 9760-9765), or styrene (Benedetti 2012  J. Am. Chem. Soc.,  134, 12418-12421) cores significantly red-shift the excitation and emission properties, and in the case of the latter two, improve brightness through improvements in their excitation peak extinction coefficients. The entire content of each of the references cited above (as well as all other references referred to herein including the contents of nucleic acid and amino acid sequence accession number references) are incorporated herein by reference. Non-limiting examples of prodan analogues include 2-cyano-6-dihexylaminoanthracene and 2-propionyl-6-dihexylaminoanthracene, as well as fluorophores comprising the following structures: 
     
       
         
         
             
             
         
       
     
     In some embodiments, the fluorophore comprises a fluorescent protein. Fluorescent proteins that emit blue, cyan, green, yellow, orange, red, far-red, or near infrared radiation when contacted with excitation radiation are known in the art and commercially available as proteins and via the expression of vectors that encode the fluorescent protein. Non-limiting examples of fluorescent proteins include Blue Fluorescent Protein (BFP), TagBFP. mTagBFP2, Azurite, Enhanced Blue Florescent Protein 2 (EBFP2), mKalama1, Sirius, Sapphire, T-Sapphire, Cyan Fluorescent Protein (CFP); Enhanced Cyan Fluorescent Protein (ECFP), Cerulean. SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFP1, AmCyan1, Green Fluorescent Protein (GFP), Enhanced Green Fluorescent Protein (EGFP), Emerald, Superfolder GFP, AcGFP1. ZsGreen1, Monomeric Azami Green, TagGFP2. mUKG, mWasabi, Clover, mNeonGreen. Yellow Fluorescent Protein (YFP), Enhanced Yellow Fluorescent Protein (EYFP), Citrine, Venus, Super Yellow Fluorescent Protein 2 (SYFP2), TagYFP, ZsYellow1, mBanana, Orange Fluorescent Protein (OFP), Monomeric Kusabira-Orange (mKO), mKOκ, mKO2, mOrange, mOrange2, Red Fluorescent Protein (RFP), DsRed-Express, DsRed-Express2, DsRed2, AsRed2, mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mRuby2, mPlum, HcRed-Tandem, mKate2, mNeptune, HcRed1, E2-Crimson, NirFP, TagRFP657, IFP1.4, or iRFP. 
     In some embodiments, the fluorophore comprises a quantum dot (Medintz et al. 2005) (Sapsford, Berti and Medintz 2006 Angew Chem Int Ed Engl, 45, 4562-89; Resch-Genger et al. 2008 Nat Methods, 5, 763-75). In some embodiments the emission properties of the conjugated protein are enhanced by immobilization on or near metallic nanoparticles (Zeng et al. 2014  Chem Soc Rev,  43, 3426-52; Shen et al. 2015  Nanoscale,  7, 20132-41). 
     In various embodiments, the peak emission wavelength and/or the emission intensity of the biosensor change when the ligand binds to the ligand-binding protein. In some embodiments, the biosensor exhibits a dichromatic signaling change when the ligand binds to the ligand-binding protein. In various embodiments, the peak emission wavelength of the biosensor shifts by at least about 5, 10, 15, 20, 30, 40, 50, or by about 5-50 nm when the biosensor binds to ligand. In certain embodiments, the emission intensity of the biosensor increases by at least about 5%, 10%, 20%, 30%, 40%0i, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200, or 300% when the biosensor binds to ligand. In various embodiments, the signal produced by the reporter group persists for at least 1 nanoseconds (ns), 5 ns, 10 ns, 25 ns, 50 ns, 75 ns, 100 ns, 200 ns, 300 ns, 400 ns, 500 ns, 600 ns, 700 ns, 800 ns, 900 ns, 0.001 milliseconds (ms), 0.01 ms, 0.1 ms, 1 ms, 5 ms, 10 ms, 20 ms, 25 ms, 50 ms, 100 ms, or 500 ms when the ligand binds to the ligand-binding protein. 
     Ratiometric Sensing with Fluorescence Energy Transfer 
     The present subject matter provides methods for converting monochromatic responses into dichromatic responses that enable ratiometric sensing. If the fluorescence emission spectrum changes shape in response to analyte binding such that the ratio of emission intensities at two appropriately chosen wavelengths reports on analyte concentration (dichromatic response), then ratiometric measurements can be used to monitor analyte concentrations. In embodiments, these methods are based on establishing non-geometrically modulated Förster resonance energy transfer (ngmFRET) between a fluorophore (a directly responsive partner), and a second fluorophore that neither interacts directly with the ligand. nor is sensitive to ligand-mediated changes in its environment (an indirectly responsive partner). Biosensors that undergo ngmFRET (or altered ngmFRET) upon ligand binding are also provided herein, as well as compositions and devices comprising such biosensors. 
     Methods, compounds, and compositions provided herein overcome challenges regarding the design of biosensors that produce a ratiometric signal. For example, a biosensor that exhibits a monochromatic response (which does not produce a ratiometric signal) to ligand binding may be converted into a biosensor that produces a dichromatic/ratiometric signal. Moreover, the number of fluorophores that may be utilized in ratiometric biosensors is dramatically increased by the present subject matter. For example, fluorophores that typically do not show a dichromatic response to ligand binding (such as fluorescein and derivatives thereof) may be used together with an additional reporter group (such as another fluorophore) to produce a ratiometric signal. Also included are methods, compounds, and compositions relating to biosensors with multiple reporter groups that have improved ratiometric signals compared to other ratiometric biosensors (e.g., ratiometric biosensors having a single reporter group). 
     Traditional/conventional geometrically-modulated Fluorescence Resonance Energy Transfer (tgmFRET) is a physical phenomenon that was first described over 50 years ago. In tgmFRET, the transfer of excited state energy from a donor fluorophore to an acceptor fluorophore (i.e. energy transfer) is modulated by a ligand-binding event through changes in the distance and/or angle between the donor and acceptor fluorophores, tgmFRET is manifested by opposing changes in the fluorescence emission intensities of the donor and acceptor fluorophores, respectively, in response to ligand binding. For instance, a decrease in distance results in a decrease of the donor fluorescence emission intensity and an increase in the acceptor fluorescence intensity, as energy is transferred from the former to the latter. A ligand-mediated increase in the distance between the partners has the opposite effect (the fluorescence emission intensity of the donor increases, whereas that of the acceptor decreases). In tgmFRET, ligand-mediated modulation of fluorescence intensity arises from global changes in the entire system, and can occur only if both partners are present. 
     By contrast, in ngmFRET ligand-mediated modulation of fluorescence intensity arises from changes that are localized to the photophysics of the directly responsive fluorophore. Unlike tgmFRET, ligand-mediated changes in fluorescence therefore occur also if only the directly responsive partner is present in isolation by itself. Although the entire ngmFRET system comprising two partners is not required for evincing ligand-mediated changes in fluorescence emission intensity, the response of such a system is qualitatively changed or quantitatively enhanced over the responses of the isolated directly responsive partner (e.g. converting a monochromatic into a dichromatic response, thereby enabling ratiometry). Furthermore, unlike tgmFRET, the pattern of fluorescence intensity changes manifested by ligand binding in ngmFRET systems are not limited to opposing changes only. Instead, in ngmFRET almost all combinations of emission intensity changes are possible: opposing changes in the two partners, both partners increase, both decrease, one partner remains unchanged whereas the other increases or decreases. The majority of these responses evince changes that are unequal in magnitude and/or direction (i.e. increase, decrease), and accordingly are manifested as ligand-mediated changes in the ratio of the two fluorescence emission intensities. This versatility of ngmFRET system response patterns has great utility in the field of fluorescent biosensors. 
     The ligand-mediated alteration of the photophysics of the directly responsive partner includes changes to its spectral properties such as the shape of the excitation or emission spectra, and the ratio of radiative to non-radiative emission rates. The fluorescence emission intensity of the indirectly responsive partner in isolation does not change in response to ligand binding; its intensity changes only in the presence of a directly responsive partner in the complete ngmFRET system. In the field fluorescence spectroscopy, the term “quenching” has often been used loosely to refer to a decrease fluorescence emission intensity. However, as used herein, the term “quenching” strictly means a “change in the ratio of radiative to non-radiative emission rates” of a fluorophore. 
     Aspects of the present subject matter provide biosensors in which ngmFRET occurs between two or more reporter groups (e.g., a donor fluorophore and an acceptor fluorophore) of the biosensor. For example, ngmFRET may change (e.g., increase or decrease) when ligand is bound to the biosensor and a donor fluorophore is contacted with radiation within its excitation wavelength. Effects from tgmFRET and ngmFRET may occur together and be combined into an overall ligand-mediated change in fluorescence emission intensity. In preferred embodiments, less than half or none of the change in overall ligand-mediated change in fluorescence emission intensity is due to tgmFRET. In embodiments, most of the overall ligand-mediated change in fluorescence emission intensity change is not due to a change in the distance between the donor and acceptor fluorophore or as a result of a change in the orientation between the donor and acceptor fluorophore. In non-limiting examples, less than about 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0.5% of the change in overall ligand-mediated change in fluorescence emission intensity is due to tgmFRET. In various embodiments, at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 99.99% of the ligand-mediated change in fluorescence emission intensity is due to ngmFRET. For example, the change in overall ligand-mediated change in fluorescence emission intensity comprises a spectral change (e.g., in the excitation or emission spectrum) and/or a change in the ratio of the radiative to non-radiative decay rates of one of the fluorophores (by itself and regardless of the presence of any other fluorophore/partner) upon ligand binding. 
     In some embodiments, ligand binding mediates spectral shifts in the absorption or emission spectrum of the directly responsive partner. In certain embodiments such changes are due at least in part to a switch between different excited states in the ligand-free and ligand-bound biosensor. The two excited states are associated with different transition dipoles. This class of changes is termed “dipole switching” herein. 
     In embodiments, the reporter groups include a directly responsive partner (which may be a donor fluorophore or an acceptor fluorophore) and an indirectly responsive partner (which may be a donor fluorophore or an acceptor fluorophore). Depending on context, a “directly responsive” partner is a fluorophore that responds to (i) ligand-induced protein conformational changes upon ligand binding to a ligand-binding protein: or (ii) ligand binding to the directly responsive partner itself. In some embodiments, the directly responsive partner comprises a fluorophore (i.e., it is a directly responsive fluorophore). In various embodiments, the directly responsive fluorophore exhibits a monochromatic or dichromatic spectral change, and/or a change in the ratio of radiative to non-radiative emission rates, upon ligand binding. In certain embodiments relating to ligand binding to the directly responsive partner itself, the directly responsive partner may be a fluorophore such as a fluorescent protein or a small molecule fluorescent compound. An “indirectly responsive” partner is a fluorophore for which no change in emission spectra, excitation spectra, or change in the ratio of radiative to non-radiative emission rates is caused by ligand binding in the absence of a directly responsive partner. In some embodiments, the indirectly responsive partner comprises a fluorophore (i.e., it is an indirectly responsive fluorophore). When paired with a directly responsive partner with which the indirectly responsive partner is a ngmFRET donor or acceptor, the emission fluorescence intensity of the indirectly responsive partner changes due to a change in energy flow in the ngmFRET pathway upon ligand binding. See, e.g.,  FIG. 74 . 
     ngmFRET Biosensors 
     Provided herein are methods, compositions, biosensors, and devices comprising multiple reporter groups, e.g. a directly responsive fluorophore and an indirectly responsive fluorophore, between which ngmFRET occurs. 
     Aspects include a method of detecting a ligand in a sample, comprising contacting a biosensor with a ligand. In various embodiments, the ligand comprises bicarbonate or Ca 2+ . The biosensor comprises a ligand-binding protein, a directly responsive fluorophore and an indirectly responsive fluorophore. The directly responsive and the indirectly responsive fluorophores are located at two distinct sites of the ligand-binding-protein. In some embodiments, the directly responsive fluorophore is a donor fluorophore and the indirectly responsive fluorophore is an acceptor fluorophore. Alternatively, the directly responsive fluorophore is an acceptor fluorophore and the indirectly responsive fluorophore is a donor fluorophore. The method includes contacting the biosensor with radiation comprising a wavelength within the excitation spectrum of the donor fluorophore. When the biosensor is contacted with such radiation, a fluorescence property of the directly responsive fluorophore changes in response to ligand binding. This change in fluorescent property is independent of the indirectly responsive fluorophore, and occurs regardless of whether the indirectly responsive fluorophore is absent or present. The fluorescence properties of the indirectly responsive fluorophore do not change in response to ligand binding in the absence of the directly responsive fluorophore. When the biosensor is contacted with radiation comprising a wavelength within the excitation spectrum of the donor fluorophore, then (i) ngmFRET occurs between the directly responsive fluorophore and the indirectly responsive fluorophore; (ii) fluorescent light is emitted from the biosensor, and the light emitted from the biosensor comprises a combination of light emitted from the directly responsive fluorophore and light emitted from the indirectly responsive fluorophore; and (iii) the ratio of the fluorescence emission intensity emitted from the biosensor at each of two distinct wavelengths changes in response to ligand binding. In various embodiments, the method further comprises measuring fluorescent light that is emitted from the directly responsive fluorophore and the indirectly responsive fluorophore, and calculating a ratiometric signal to detect the ligand in the sample. 
     The ratiometric signal (R 1,2 ) comprises a quotient of two intensities, I λ1  and I λ2 . measured at two independent wavelengths, λ 1  and λ 2  and is calculated according to the following equation: 
         R   1,2   =I   λ1   /I   λ2 . 
     The two independent wavelengths λ 1  and λ 2  may be from a single fluorophore or from a combination of two or more fluorophores (e.g., a pair of fluorophores between which ngmFRET occurs). In some embodiments, λ 1  falls within the emission spectrum of a directly responsive fluorophore and λ 2  falls within the emission spectrum of an indirectly responsive fluorophore. In certain embodiments. λ 1  falls within the emission spectrum of an indirectly responsive fluorophore and λ 2  falls within the emission spectrum of a directly responsive fluorophore. In various embodiments, λ 1  falls within the emission spectrum of both a directly responsive fluorophore and an indirectly responsive fluorophore. In various embodiments, λ 2  falls within the emission spectrum of both a directly responsive fluorophore and an indirectly responsive fluorophore. 
     Aspects of the present subject matter provide FRSs whose emission spectra change (e.g., the shape of the emission spectra change) in response to ligand binding. In various embodiments, the ratio of intensities at two chosen wavelengths of an FRS&#39;s emission spectrum changes upon ligand binding. 
     In various embodiments, the emission spectra of two or more fluorophores contributes to I λ1  and/or I λ2 . In some embodiments, the emission spectrum of a directly responsive fluorophore contributes to I λ1  and/or I λ2  and the emission spectrum of an indirectly responsive fluorophore contributes to I λ1  and/or I λ2 . In certain embodiments, a directly responsive fluorophore contributes to I λ1  and the emission spectrum of an indirectly responsive fluorophore contributes to I λ2 . In some embodiments, a directly responsive fluorophore contributes to I λ2  and the emission spectrum of an indirectly responsive fluorophore contributes to I λ1 . In various embodiments, both the emission spectrum of a directly responsive fluorophore and the emission spectrum of an indirectly responsive fluorophore contributes to I λ1 . In some embodiments, both the emission spectrum of a directly responsive fluorophore and the emission spectrum of an indirectly responsive fluorophore contributes to I λ2 . 
     In some embodiments, the directly responsive fluorophore is Pacific Blue and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, or 460 nm), and wherein the indirectly responsive fluorophore is Oregon Green and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, or 540 nm). In some embodiments, the directly responsive fluorophore is Pacific Blue and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, or 470 nm), and wherein the indirectly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, or 540 nm). In some embodiments, the directly responsive fluorophore is Badan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 470, 471, 472, 473, 474, 475, 476, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, or 505 nm), and wherein the indirectly responsive fluorophore is Alexa532 and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, or 570 nm). In some embodiments, the directly responsive fluorophore is Badan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, or 570 nm), and wherein the indirectly responsive fluorophore is Alexa532 and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, or 510 nm). In some embodiments, the directly responsive fluorophore is Badan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, or 570 nm), and wherein the indirectly responsive fluorophore is Alexa532 and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 470, 471, 472, 473, 474, 475, 476, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, or 505 nm). In some embodiments, the directly responsive fluorophore is Badan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g. including a wavelength of about 450, 451, 452, 453, 454, 454, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, or 475 nm), and wherein the indirectly responsive fluorophore is Alexa532 and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, or 570 nm). In some embodiments, the directly responsive fluorophore is Pacific Blue and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, or 460 nm), and wherein the indirectly responsive fluorophore is Texas Red and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, or 670 nm). In some embodiments, the directly responsive fluorophore is Pacific Blue and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, or 460 nm), and wherein the indirectly responsive fluorophore is Texas Red and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, or 670 nm). In some embodiments, the directly responsive fluorophore is Pacific Blue and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, or 460 nm). and wherein the indirectly responsive fluorophore is Texas Red and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, or 455 nm). In some embodiments, the directly responsive fluorophore is Acrylodan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 470, 471, 472, 473, 474, 475, 476, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, or 505) nm, and wherein the indirectly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 450, 451, 452, 453, 454, 454, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, or 475 nm). In some embodiments, the directly responsive fluorophore is Acrylodan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 470, 471, 472, 473, 474, 475, 476, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, or 505 nm). and wherein the indirectly responsive fluorophore is Alexa532 and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 450, 451, 452, 453, 454, 454, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, or 475 nm). In some embodiments, the directly responsive fluorophore is Alexa532 and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, or 570) nm, and wherein the indirectly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, or 540 nm). In some embodiments, the directly responsive fluorophore is Alexa532 and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, or 570) nm, and wherein the indirectly responsive fluorophore is Oregon Green and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, or 540 nm). In some embodiments, the directly responsive fluorophore is Badan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, or 540 nm), and wherein the indirectly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g. including a wavelength of about 450, 451, 452, 453, 454, 454, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, or 475 nm). In some embodiments, the directly responsive fluorophore is Acrylodan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, or 540) nm, and wherein the indirectly responsive fluorophore is 5-IAF and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 450, 451, 452, 453, 454, 454, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, or 475 nm. In some embodiments, the directly responsive fluorophore is Acrylodan and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, or 540 nm), and wherein the indirectly responsive fluorophore is Alexa532 and emission intensity is measured at a wavelength or range of wavelengths between about 400 nm and 1000 nm (e.g., including a wavelength of about 450, 451, 452, 453, 454, 454, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, or 475 nm). 
     In various embodiments, the ligand-binding protein comprises a cysteine at the position of its amino acid sequence that aligns with position 16, 17, 18, 190, or 194 of avBicarbBP5 (SEQ ID NO: 19 or 79) when the amino acid sequence of the ligand-binding protein is aligned with the amino acid sequence of avBicarbBP5 using the ClustalW alignment program, and wherein the Pacific Blue, the Alexa532, the Acrylodan, or the Badan is covalently attached to the cysteine. In some embodiments, the 5-IAF, the Alexa532, the Texas Red, the IANBD, or the Oregon Green is attached to the N-terminus or the C-terminus of the ligand-binding protein via a fluorophore attachment motif. In a non-limiting example, the ligand-binding protein comprises an amino acid sequence of SEQ ID NO: 64. 
     In various embodiments, the change in the fluorescent property of the directly responsive fluorophore comprises (i) a bathochromic or hypsochromic shift in the emission or excitation spectrum thereof; and/or (ii) a change in the ratio of radiative to non-radiative emission rates thereof. 
     In embodiments, the directly responsive fluorophore comprises a donor fluorophore and the indirectly responsive fluorophore comprises an acceptor fluorophore. In some embodiments, the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore decreases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensities of the donor fluorophore and the acceptor fluorophore both decrease upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore increases, decreases, or remains about the same and the emission intensity of the acceptor fluorophore decreases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensities of the donor fluorophore and the acceptor fluorophore both increase upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore increases, decreases, or remains about the same and the emission intensity of the acceptor fluorophore increases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. 
     In embodiments the directly responsive fluorophore comprises an acceptor fluorophore and the indirectly responsive fluorophore comprises a donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore remains about the same and the emission intensity of the acceptor fluorophore decreases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore increases, decreases, or remains about the same upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore remains about the same and the emission intensity of the acceptor fluorophore increases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore decreases and the emission intensity of the acceptor fluorophore increases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. In some embodiments, the emission intensity of the donor fluorophore increases and the emission intensity of the acceptor fluorophore remains about the same, increases, or decreases upon ligand binding to the ligand-binding protein when the donor fluorophore is contacted with radiation within the excitation spectrum of the donor fluorophore. 
     In instances in which an emission intensity increases, the increase may be, e.g., between about 0.1% to 10%, 10% to 50%, or 50% to 100%, or at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold. In instances in which an emission intensity decreases, the decrease may be, e.g., a decrease of between about at least about 0.1% to 10%, 10% to 50%, or 50% to 00%, or at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%. In various embodiments in which both the emission intensity of the donor fluorophore and the acceptor fluorophore increases, then the increases are not equal. In certain embodiments in which both the emission intensity of the donor fluorophore and the acceptor fluorophore decreases, then the decreases are not equal. 
     In certain embodiments, the indirectly responsive fluorophore is attached to the ligand-binding protein via a covalent bond. Various approaches for attaching reporter groups such as directly and indirectly responsive fluorophores to a polypeptide such as a ligand-binding protein are described herein. In some embodiments, the covalent bond comprises a disulfide bond, a thioester bond, a thioether bond, an ester bond, an amide bond, or a bond that has been formed by a click reaction. 
     In some embodiments, the indirectly responsive fluorophore is attached to the ligand-binding protein via a non-covalent bond. In certain embodiments, the indirectly responsive fluorophore is attached to a cysteine or a lysine of the ligand-binding protein. 
     In various embodiments, the indirectly responsive fluorophore is attached to the N-terminus or the C-terminus of the protein. In some embodiments, the indirectly responsive fluorophore is attached to the N-terminus or the C-terminus of the protein via a fluorophore attachment motif. 
     In some embodiments, fluorophore attachment motif comprises a polypeptide. Various embodiments may be used to link a fluorophore with a ligand-binding protein. In some embodiments, the polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids. In a non-limiting example, the polypeptide comprises amino acids in the sequence of βZif (SEQ ID NO: 90). In another non-limiting example, the polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids in a sequence that is at least about 85%, 90%, 95%, or 99% identical to the amino acid sequence of  E. coli  thioredoxin (ecTRX; SEQ ID NO: 169). 
     In some embodiments, the directly responsive fluorophore is attached to the ligand-binding protein via a covalent bond. In various embodiments, the covalent bond comprises a disulfide bond, a thioester bond, a thioether bond, an ester bond, an amide bond, or a bond that has been formed by a click reaction. In directly responsive fluorophore is attached to a cysteine or a lysine of the protein. 
     In some embodiments, an overlap of the emission spectrum of the donor fluorophore and the excitation spectrum of the acceptor fluorophore increases upon ligand binding. In certain embodiments, the directly responsive fluorophore comprises the donor fluorophore. and the increase results from a bathochromic shift in the emission spectrum of the donor fluorophore. Alternatively, the directly responsive fluorophore comprises the acceptor fluorophore, and the increase results from a hypsochromic shift in the excitation spectrum of the acceptor fluorophore. 
     In various embodiments, an overlap of the emission spectrum of the donor fluorophore and the excitation spectrum of the acceptor fluorophore decreases upon ligand binding. In some embodiments, the directly responsive fluorophore comprises the donor fluorophore. and the decrease results from a hypsochromic shift in the emission spectrum of the donor fluorophore. In certain embodiments, the directly responsive fluorophore comprises the acceptor fluorophore, and the decrease results from a bathochromic shift in the excitation spectrum of the acceptor fluorophore. 
     In some embodiments, the directly responsive fluorophore has a monochromatic spectral change upon ligand binding. Alternatively, the directly responsive fluorophore has a dichromatic spectral change upon ligand binding. 
     In certain embodiments, the emission intensity of the donor fluorophore and/or the acceptor fluorophore increases in two phases as ligand concentration increases. 
     In various embodiments, the ratio of radiative to non-radiative emission or intensity of the directly responsive fluorophore increases by at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold upon ligand binding to the ligand-binding protein. Alternatively, the ratio of radiative to non-radiative emission or intensity of the directly responsive fluorophore decreases by at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 50%, 75%, 90%, 95%, or 99% upon ligand binding to the ligand-binding protein. 
     In embodiments, the directly responsive fluorophore and the indirectly responsive fluorophore are not a naphthalene derivative. In some embodiments, the directly responsive fluorophore and the indirectly responsive fluorophore are not Prodan, Acrylodan, or Badan. In certain embodiments, the directly responsive fluorophore is not a naphthalene derivative. In some embodiments, the directly responsive fluorophore is not Prodan, Acrylodan, or Badan. 
     In various embodiments, the directly responsive fluorophore comprises xanthene, a xanthene derivative, fluorescein, a fluorescein derivative, coumarin, a coumarin derivative, cyanine, a cyanine derivative, rhodamine, a rhodamine derivative, phenoxazine, a phenoxazine derivative, squaraine, a squaraine derivative, coumarin, a coumarin derivative, oxadiazole, an oxadiazole derivative, anthracene, an anthracene derivative, a boradiazaindacine (BODIPY) family fluorophore, pyrene, a pyrene derivative, acridine, an acridine derivative, arylmethine, an arylmethine derivative, tetrapyrrole, or a tetrapyrrole derivative. In some embodiments, the directly responsive fluorophore comprises fluorescein or a derivative thereof. 
     In some embodiments, the directly responsive fluorophore and/or the indirectly responsive fluorophore comprises a fluorescent protein. In various embodiments, the directly responsive fluorophore and/or the indirectly responsive fluorophore comprises an organic compound having a molecular weight less than about 2000 Da (e.g., 5-iodoacetamidofluorescein (5-IAF) or 6-iodoacetamidofluorescein (6-IAF), rhodamine, Oregon Green, eosin, Texas Red, indocarbocyanine, oxacarbocyanine, thiacarbocyanine. merocyanine, Badan, Acrylodan, IAEDANS, comprising 3-cyano-7-hydroxycoumarin, 7-hydroxycoumarin-3-carboxylic acid, 6,8-difluoro-7-hydroxy-4-methylcoumarin, or 7-amino-4-methylcoumarin, pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole. DRAQ5, DRAQ7. or CyTRAK Orange, cascade blue. Nile red, Nile blue, cresyl violet, oxazine 170. proflavin, acridine orange, acridine yellow, auramine, crystal violet, malachite green, porphin, phthalocyanine, bilirubin, pyrene, N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-ox-a-1,3-diazol-4-yl)ethylenediamine (NBD), N-((2-(iodoacetoxy)ethyl)-N-methy-1)amino-7-nitrobenz-2-oxa-1,3-diazole (NBDE), JPW4039, JPW4042, JPW4045, Pacific Blue, CPM, N,N′-Dimethyl-N-(Iodoacetyl)-N′-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Ethylenediamine (IANBD), 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM), BODIPY 499, BODIPY 507/545, BODIPY 499/508, Alexa 432, Alexa488, Alexa532, Alexa546, Cy5, or 1-(2-maleimidyl ethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium methanesulfonate (PyMPO maleimide) (PyMPO)). Numerous combinations of directly responsive fluorophores and indirectly responsive fluorophores are possible. For example, in various non-limiting examples, (a) the donor fluorophore comprises Pacific Blue and the acceptor fluorophore comprises 5-IAF or 6-iodoacetamidofluorescein (6-IAF); (b) the donor fluorophore comprises Pacific Blue and the acceptor fluorophore comprises Oregon Green; (c) the donor fluorophore comprises IAEDANS and the acceptor fluorophore comprises 5-IAF or 6-IAF; (d) the donor fluorophore comprises acrylodan and the acceptor fluorophore comprises Alexa532; (e) the donor fluorophore comprises acrylodan and the acceptor fluorophore comprises 5-IAF or 6-IAF; (f) the donor fluorophore comprises acrylodan and the acceptor fluorophore comprises Pacific Blue or YFP; (g) the donor fluorophore comprises 5-IAF or 6-IAF and the acceptor fluorophore comprises Pacific Blue: (h) the donor fluorophore comprises badan and the acceptor fluorophore comprises 5-IAF or 6-IAF; or (i) the donor fluorophore comprises badan and the acceptor fluorophore comprises Alexa532. 
     Aspects also include a biosensor for a ligand comprising a ligand-binding protein, a directly responsive fluorophore and an indirectly responsive fluorophore, the directly responsive and the indirectly responsive fluorophores being located at two distinct sites of the ligand-binding-protein, wherein (i) the directly responsive fluorophore is a donor fluorophore and the indirectly responsive fluorophore is an acceptor fluorophore; or (ii) the directly responsive fluorophore is an acceptor fluorophore and the indirectly responsive fluorophore is an donor fluorophore. In various embodiments, the ligand comprises bicarbonate or Ca 2+ . 
     Any of the ligand-binding proteins disclosed herein, as well as others, may be included in the biosensors and methods that are provided. 
     Aspects of the present subject matter also provide a method for constructing a biosensor, comprising: (a) providing a ligand-binding protein; (b) identifying at least one putative allosteric, endosteric, or peristeric site of the ligand-binding based a structure of the ligand-binding protein; (c) mutating the ligand-binding protein to substitute an amino acid at the at least one putative allosteric, endosteric, or peristeric site of the second protein with a cysteine; (d) conjugating a donor fluorophore or an acceptor fluorophore to the cysteine to produce single labeled biosensor; (e) detecting whether there is a spectral shift or change in emission intensity of the single labeled biosensor upon ligand binding when the donor fluorophore or the acceptor fluorophore is fully excited; and (f) if a spectral shift or change in emission intensity is detected in (e), attaching a donor fluorophore to the second protein if an acceptor fluorophore is attached to the cysteine, and attaching an acceptor fluorophore to the second protein if an acceptor fluorophore is attached to the cysteine. 
     In various embodiments, the ligand-binding protein has been identified by (i) selecting a first protein having a known amino acid sequence (seed sequence), wherein the first protein is known to bind a ligand; (ii) identifying a second protein having an amino acid sequence (hit sequence) with at least 15% sequence identity to the seed sequence; (iii) aligning the seed amino acid sequence and the hit sequence, and comparing the hit sequence with the seed sequence at positions of the seed sequence that correspond to at least 5 primary complementary surface (PCS) amino acids, wherein each of the at least 5 PCS amino acids has a hydrogen bond interaction or a van der Waals interaction with ligand when ligand is bound to the first protein; and (iv) identifying the second protein to be a ligand-binding protein if the hit sequence comprises at least 5 amino acids that are consistent with the PCS. 
     In some embodiments, the spectral shift comprises a monochromatic fluorescence intensity change or a dichromatic spectral shift. 
     Also provided is a method of converting a biosensor that shows a monochromatic response upon ligand binding into a biosensor with a dichromatic response upon ligand binding, the method comprising (a) selecting a biosensor that exhibits a monochromatic response upon ligand binding, wherein the biosensor comprises a ligand-binding protein and a first reporter group; and (b) attaching a second reporter group to the biosensor, wherein the second reporter group has (i) an excitation spectrum that overlaps with the emission spectrum of the first reporter group; or (ii) an emission spectrum that overlaps with the excitation spectrum of the first reporter group. 
     Also provided is a method of increasing a dichromatic response of a biosensor to ligand binding, the method comprising (a) selecting a biosensor that exhibits a dichromatic response upon ligand binding, wherein the biosensor comprises a ligand-binding protein and a first reporter group; and (b) attaching a second reporter group to the biosensor, wherein the second reporter group has (i) an excitation spectrum that overlaps with the emission spectrum of the first reporter group; or (ii) an emission spectrum that overlaps with the excitation spectrum of the first reporter group. 
     In some embodiments, the second reporter group is within about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 4, 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, or 200 angstroms (Å) of the first reporter group regardless of whether ligand is bound to the biosensor. Suitable distances may be determined in part by the distance-dependence of the energy transfer between a given donor-acceptor pair (see, e.g. J. R Lakowicz, 2006, Principles of Fluorescence Spectroscopy, Springer, incorporated herein by reference). In some embodiments, when the ligand is bound to the biosensor, the average distance between the first reporter group and the second reporter group changes by less than about 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.01 angstroms (Å) compared to when ligand is not bound to the ligand-binding protein. 
     In various embodiments, if the acceptor fluorophore comprises palladium, platinum, ruthenium, or osmium, then the acceptor fluorophore is not attached to the amino group of the N-terminus of the ligand-binding protein. In some embodiments, the acceptor fluorophore does not comprise [Ru(bpy) 3 ] 2+ . [Ru(Ph 2 phen) 3 ] 2+ , [Ru(bpy) 2 (dcbpy)] 2+ , or [Ru(bpy) 2 (phen-ITC)] 2+ . where bpy is 2,2′-bipyridine, phen is 1,10-phenanthroline. dcbpy is 4,4′-dicarboxy-2,2′-bipyridine, and ITC is isothiocyanate. In certain embodiments, the biosensor does not comprise an  E. coli  glutamine-binding protein with Acrylodan attached to 179C. In some embodiments, the biosensor does not comprise  E. coli  ligand-binding protein with Acrylodan attached to 255C. 
     tgmFRET Biosensors 
     While ngmFRET is preferred to tgmFRET, tgmFRET may be used alternatively or in addition to ngmFRET in certain embodiments. 
     In various embodiments, the biosensor comprises multiple reporter groups, including a first reporter group and a second reporter group. For example, the first reporter group may comprise a donor fluorophore and the second reporter group may comprise an acceptor fluorophore. In certain embodiments, FRET is detectable by a change in the fluorescence of the acceptor fluorophore or by a decrease in of donor fluorophore fluorescence. In various embodiments, the donor fluorophore, and/or the acceptor fluorophore is fluorescent. In some embodiments, both the donor fluorophore and the acceptor fluorophore are fluorescent. 
     In various embodiments, the angle and/or distance between the donor fluorophore and the acceptor fluorophore changes upon ligand binding. In some embodiments, neither the donor fluorophore nor the acceptor fluorophore is directly responsive to ligand binding. In some embodiments the donor fluorophore and/or the acceptor fluorophore is attached to the N-terminus or the C-terminus of the ligand-binding protein (e.g., directly or via a fluorophore attachment motif). In certain embodiments, the donor fluorophore and/or the acceptor fluorophore is attached to a fluorophore attachment motif. For example, the fluorophore attachment motif may be conjugated to the N-terminus or the C-terminus of the ligand-binding protein. 
     In some embodiments, the donor fluorophore and/or the acceptor fluorophore comprises a fluorescent protein. In various embodiments, the donor fluorophore and/or the acceptor fluorophore comprises an organic compound having a molecular weight less than about 2000 Da (e.g., 5-iodoacetamidofluorescein (5-IAF) or 6-iodoacetamidofluorescein (6-IAF), rhodamine, Oregon Green, eosin. Texas Red. indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine, Badan, Acrylodan, IAEDANS, comprising 3-cyano-7-hydroxycoumarin, 7-hydroxycoumarin-3-carboxylic acid, 6,8-difluoro-7-hydroxy-4-methylcoumarin, or 7-amino-4-methylcoumarin. pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole. DRAQ5, DRAQ7, or CyTRAK Orange, cascade blue, Nile red, Nile blue, cresyl violet, oxazine 170, proflavin, acridine orange, acridine yellow, auramine, crystal violet, malachite green, porphin, phthalocyanine, bilirubin, pyrene, N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-ox-a-1,3-diazol-4-yl)ethylenediamine (NBD), N-((2-(iodoacetoxy)ethyl)-N-methy-1)amino-7-nitrobenz-2-oxa-1,3-diazole (NBDE), Acrylodan, JPW4039, JPW4042. JPW4045, Oregon Green, Pacific Blue, CPM, N,N′-Dimethyl-N-(Iodoacetyl)-N′-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Ethylenediamine (IANBD), 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM), BODIPY 499, BODIPY 507/545, BODIPY 499/508, Alexa 432, Alexa488, Alexa532, Alexa546, Cy5, or 1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridinium methanesulfonate (PyMPO maleimide) (PyMPO)). For example, the organic compound is a fluorophore. Numerous combinations of donor and acceptor fluorophores are possible. 
     Fluorophore Attachment Motifs 
     Aspects of the present subject matter include the use of one or more fluorophore attachment motifs to attach one or more reporter groups to a ligand-binding protein. For example, a reporter group may be attached to a fluorophore attachment motif that is attached to the N-terminus or the C-terminus of the ligand-binding protein. 
     In various implementations, the fluorophore attachment motif comprises a polypeptide. In some embodiments, the polypeptide comprises amino acids in the βZif amino acid sequence (SEQ ID NO: 90). 
     In some embodiments, the polypeptide comprises a stretch of at least 50, 60, 70, 80, 90, or 100 amino acids in a sequence that is at least about 85%, 90%, 95%, or 99% identical to the amino acid sequence of  E. coli  thioredoxin (ecTRX; SEQ ID NO: 169). In some embodiments, the polypeptide is a mutant of ecTRX comprising a D3X, K4X, K19X, D27X, K37X, K53X, K58X, K70X, R74X, K83X. K91X, K97X, or K101X mutation, or any combination thereof, wherein X is any amino acid, and wherein each ecTRX amino acid position is numbered as in SEQ ID NO: 169. In certain embodiments, the polypeptide is a mutant of ecTRX comprising a D3A, K4R, K4Q, K19R, K19Q, D27A, K37R, K53M, K53R, K58M, K70R, R74C, K83R, K91R, K97R, or K101R mutation, or any combination thereof, wherein each ecTRX amino acid position is numbered as in SEQ ID NO: 169. 
     In non-limiting examples, the polypeptide comprises amino acids in the sequence set forth as any one of SEQ ID NOS: 169-187. 
     In certain embodiments, the polypeptide comprises (a) at least 1, 2, or 3 thiol groups; (b) at least 1, 2, or 3 cysteines that each comprise a sulfhydryl group; (c) at least 1, 2, or 3 primary amine groups; and/or (d) at least 1, 2, or 3 lysines that each comprise a primary amine. In some embodiments there is no disulfide bond between cysteines within the amino acid sequence of the polypeptide. 
     In some embodiments, the polypeptide comprises a hexahistidine tag. In some embodiments, the hexahistidine tag is attached to another portion of the polypeptide via a GGS linker. 
     Exemplary Methods of Using Biosensors Provided Herein 
     Aspects of the present subject matter provide a method of assaying for a ligand in a sample. The method may include contacting the sample with a biosensor disclosed herein under conditions such that the ligand-binding protein of the biosensor binds to the ligand if ligand is present in the sample. The method also comprises detecting (i) whether a signal is produced by a reporter group of the biosensor; and/or (ii) the a signal produced by a reporter group of the biosensor. In a non-limiting example, a reporter group of the biosensor is fluorescent, and the method further comprises contacting the reporter group with electromagnetic radiation having a wavelength that comprises a wavelength within the band of excitation wavelengths of the reporter group. 
     In various embodiments, the method further comprises (i) comparing a signal produced by a reporter group of the biosensor when the biosensor is contacted with the sample with a signal produced by a control sample containing a known quantity of ligand (e.g., ligand at a concentration of about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, or 150 mM, or a series of control samples having concentrations within the range of about 0.5 mM to about 150 mM); and (ii) detecting the presence or absence of ligand in the sample based on this comparison. In embodiments the control sample lacks ligand (e.g. the concentration of ligand is 0 mM). Alternatively or in addition, the method further comprises (i) comparing a signal produced by a reporter group of the biosensor when the biosensor is contacted with the sample with signals produced by a series of control samples containing known quantities of ligand; and (ii) determining the quantity of ligand in the sample based on this comparison. In some embodiments, the series of control samples comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 control samples, and wherein each control sample comprises a different quantity of ligand. Alternatively or in addition, the method further comprises determining the concentration of a ligand in a sample, wherein determining the concentration of the ligand in the sample comprises comparing the signal to a standard hyperbolic ligand binding curve to determine the concentration of the ligand in the test sample, wherein the standard hyperbolic ligand binding curve is prepared by measuring the signal produced by the reporter group of the biosensor when the biosensor is contacted with control samples containing known concentrations of ligand. In various embodiments, the method comprises (i) measuring a ratiometric change (ΔR) and/or an intensity change (ΔI) of a signal produced by the reporter group. In some embodiments, the method includes quantitating the level of ligand present in the sample. 
     In embodiments, the ligand comprises bicarbonate and the ligand-binding protein comprises a bicarbonate-binding protein. In some embodiments, the bicarbonate-binding protein has an affinity for bicarbonate that is higher than its naturally occurring counterpart. In certain embodiments relating to the detection or measurement of bicarbonate, Ca 2+  and/or iron is added to a reaction composition, device, or sample before, at the same time as, or after a biosensor is contacted with Ca 2+  or with a sample that comprises or is being tested for Ca 2+ . In some embodiments, the Ca 2+  and/or iron is added to a concentration of at least about 0. I1 mM, 0.5 mM, 1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 2 mM, 3 mM 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM of Ca 2  and/or iron, e.g., in the sample or reaction composition. In certain embodiments, Ca 2+  and/or iron is added to a device such that it is present at a concentration of at least about 0.1 mM, 0.5 mM, 1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM when a sample or reaction mixture is added to the device, e.g., when a part/chamber of the device is filled with the sample or reaction composition. 
     In embodiments, the ligand comprises Ca 2+  and the ligand-binding protein comprises a calcium-binding protein. In some embodiments, the calcium-binding protein has an affinity for bicarbonate that is higher than its naturally occurring counterpart. In certain embodiments relating to the detection or measurement of Ca 2′ , bicarbonate is added to a reaction composition, device, or sample before, at the same time as, or after a biosensor is contacted with Ca 2+  or with a sample that comprises or is being tested for Ca 2+ . In some embodiments, the bicarbonate is added to a concentration of at least about 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 75 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, or 1000 mM of bicarbonate, e.g., in the sample or reaction composition. In certain embodiments, bicarbonate is added to a device such that it is present at a concentration of at least about 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 75 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, or 1000 mM when a sample or reaction mixture is added to the device, e.g., when a part/chamber of the device is filled with the sample or reaction composition. 
     Aspects of the present subject matter also provide a method of assaying for multiple ligands in a sample, wherein the multiple ligands comprise a first ligand and a second ligand. Such a method may include contacting the sample with (i) a first biosensor a first ligand provided herein and (ii) a second biosensor for the second ligand, under conditions such that the ligand-binding protein of the first biosensor binds to the first ligand, if the first ligand is present in the sample, and detecting (i) a signal, e.g. magnitude of the signal, produced by a reporter group of the first biosensor, or (ii) whether a signal is produced by a reporter group of the first biosensor. In some embodiments, the second biosensor is also a biosensor provided herein, and the second biosensor is contacted with the second ligand under conditions such that the ligand-binding protein of the second biosensor binds to the second ligand it is present in the sample. The method may further comprise detecting (i) a signal, e.g. magnitude of the signal, produced by a reporter group of the second biosensor, or (ii) whether a signal is produced by a reporter group of the second biosensor. 
     In some embodiments, the signal produced by the reporter group of the first biosensor is different than the signal produced by the reporter group of the second biosensor. In a non-limiting example, the reporter group of the first biosensor and the reporter group of the second biosensor are each fluorescent, and the peak emission wavelength of the reporter group of the first biosensor is at least about 10, 25, 50, 75, or 100 nm greater or lower than the peak emission wavelength of the reporter group of the second biosensor. 
     Non-limiting examples of biosensors that may be used as the second biosensor include biosensors with ligand-binding proteins comprising a GGBP (e.g., an  E. coli  GGBP) or a derivative or mutant thereof; (ii) an  E. coli  arabinose binding protein (e.g., an  E. coli  arabinose binding protein) or a derivative or mutant thereof; (iii) a dipeptide binding protein (e.g., an  E. coli  dipeptide binding protein) or a derivative or mutant thereof: (iv) a histidine binding protein (e.g., an  E. coli , histidine binding protein) or a derivative or mutant thereof; (v) a ribose binding protein (e.g., an  E. coli  ribose binding protein) or a derivative or mutant thereof; (vi) a sulfate binding protein (e.g., an  E. coli  sulfate binding protein) or a derivative or mutant thereof: (vii) a maltose binding protein (e.g., an  E. coli  maltose binding protein) or a derivative or mutant thereof; (viii) a glutamine binding protein (e.g., an  E. coli  glutamine binding protein) or a derivative or mutant thereof; (ix) a glutamate/aspartate binding protein (e.g., an  E. coli  glutamate/aspartate binding protein) or a derivative or mutant thereof; (x) a phosphate binding protein (e.g., an  E. coli  phosphate binding protein) or a derivative or mutant thereof; or (xi) an iron binding protein [e.g., a  Haemophilus  influenza ( H. influenzae ) iron binding protein] or a derivative or mutant thereof. For example, the second biosensor comprises an  E. coli  GGBP having a Y10C, Y10A, D14A, D14Q, D14N, D14S, D14T, D14E, D14H, D14L, D14Y, D14F, D14C, N15C, F16L, F16A, F16Y, F16C, N91A, K92C, E93C, S112A, S115A, E149C, E149K, E149Q, E149S, H152A, H152F, H152Q, H152N, H152C, D154A, D154C, D154N, A155S, A155H, A155L, A155F, A155Y, A155N, A155K, A155M, A155W, A155Q, A155C, R158A, R158K, R158C, M182C, M182W, W183C, W183A, N211F, N211W, N211K, N211Q, N211S, N211H, N211M, N211C, D212C, D236A, D236N, L238C, L255C, N256A, N256D, D257C, V293C, P294C, or V296C mutation (e.g., comprising 1, 2, 3, 4, 5 or more of these mutations), wherein each amino acid position is numbered as in (SEQ ID NO: 94); (ii) an  E. coli  arabinose binding protein having a D257C, F23C, K301C, L253C, or L298C mutation (e.g., comprising 1, 2, 3, 4, or 5 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681, the entire contents of which are incorporated herein by reference) (see, e.g., U.S. Patent Application Publication No. 2004/0118681, the entire contents of which are incorporated herein by reference): (iii) an  E. coli  dipeptide binding protein having a D450C, K394C, R141C, S111C, T44C, or W315C mutation (e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681, the entire contents of which are incorporated herein by reference); (iv) an  E. coli , histidine binding protein having a E167C. K229C, V163C, Y230C, F231C, Y88C mutation (e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681, the entire contents of which are incorporated herein by reference); (v) an  E. coli  ribose binding protein having a T135C, D165C, E192C, A234C, L236C, or L265C mutation (e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681, the entire contents of which are incorporated herein by reference); (vi) an  E. coli  sulfate binding protein having a L65C, N70C, Q294C, R134C, W290C, or Y67C mutation (e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference); (vii) an  E. coli  maltose binding protein having a D95C, F92C, E163C, G174C, 1329C, or S233C mutation (e.g., comprising 1, 2, 3, 4, 5 or 6 of these mutations) (see, e.g. U.S. Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference): (viii) an  E. coli  glutamine binding protein having a N160C, F221C, K219C, L162C, W220C, Y163C, or Y86C mutation (e.g., comprising 1, 2, 3, 4, 5 or more of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference): (ix) an  E. coli  glutamate/aspartate binding protein having a A207C, A210C, E119C, F126C, F131C, F270C, G211C, K268C, Q123C, or T129C mutation (e.g., comprising 1, 2, 3, 4, 5 or more of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference); (x) an  E. coli  phosphate binding protein having a A225C, N223C, N226C, S164C, or S39C mutation (e.g., comprising 1, 2, 3, 4, or 5 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference); or (xi) a  Haemophilus influenza  ( H. influenzae ) iron binding protein having a E203C, K202C, K85C, or V287C mutation (e.g., comprising 1, 2, 3, or 4 of these mutations) (see, e.g., U.S. Patent Application Publication No. 2004/0118681 the entire content of which is incorporated herein by reference). In various embodiments, the sample is suspected of comprising bicarbonate and/or Ca 2+ . 
     
       
         
           
               
            
               
                   
               
               
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     Various types of samples may be used in methods provided herein. In non-limiting examples, a sample may comprise a reaction product, a buffer, and/or a solvent. In some embodiments, the solvent is an aqueous solvent. In some embodiments, the solvent comprises a non-polar solvent, a polar aprotic solvent, and/or a polar protic solvent. For example, a sample may comprise water, liquid ammonia, liquid sulfur dioxide, sulfuryl chloride, sulfuryl chloride fluoride, phosphoryl chloride, dinitrogen tetroxide, antimony trichloride, bromine pentafluoride, hydrogen fluoride, dimethyl sulfoxide, hexane, benzene, toluene, 1,4-dioxane. chloroform, diethyl ether, dichloromethane, N-methylpyrrolidone, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, tormic acid, n-butanol, isopropanol, nitromethane, ethanol, methanol, and/or acetic acid. 
     In embodiments, a sample comprises a Newtonian liquid, a shear thickening liquid, a shear thinning liquid, a thixotropic liquid, a rheopectic liquid, or a Bingham plastic. In some implementations, a sample has a dynamic viscosity of at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, or 2 pascal-seconds (Pa·s) or less than about 2, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5 Pa·s; and/or a kinematic viscosity of at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, or 2 centistokes (cSt) or less than about 2, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5 cSt. 
     In various embodiments, the sample comprises a biological sample. The sample may comprise, e.g., a clinical sample (i.e., a sample collected in a clinical or veterinary setting, e.g., by or at the request or supervision or direction of a doctor, nurse, aid worker, or medic) and/or a physiological sample (a sample collected from an organism, e.g., a mammal such as a human). In certain embodiments, the biological sample comprises or has been provided or obtained from a skin surface or a mucosal surface. In some embodiments, the biological sample comprises a biological fluid. Non-limiting examples of biological fluids include sweat, tear fluid, blood, serum, plasma, interstitial fluid, amniotic fluid, sputum, gastric lavage, skin oil, milk, fecal matter, emesis, bile, saliva, urine, mucous, semen, lymph, spinal fluid, synovial fluid, a cell lysate, venom, hemolymph, and fluid obtained from plants such as the fluid transported in xylem cells or phloem sieve tube elements of a plant (e.g. sap). 
     The present subject matter also provides biosensors, methods, compositions, and devices useful for measuring the level of a ligand within a liquid solution or suspension or composition comprising cultured cells or tissue or a supernatant of such a solution or suspension, e.g., a sample of conditioned media or a sample of growth media in which a population of cells was cultured. In some embodiments, the sample is within a culture (e.g., inserted into a bioreactor) or provided from a media, culture, or reaction, e.g., in a bioreactor. For example, the sample may be within or provided from a fermenter such as a culture or culture supernatant from a fermentation reaction (e.g., an ongoing fermentation, the culture of cells in research settings, the production of a compound, etc.). Thus, the level of a ligand can be assayed at a timepoint of interest or at a series of timepoints over the duration of cell culture, e.g. continuously, in or from a reaction or culture. Bioreactors include devices or systems that support a biologically active environment. For example, a bioreactor may comprise a vessel in which a chemical process is carried out which involves organisms or biochemically active substances derived from such organisms. Such a process can either be aerobic or anaerobic. Organisms growing in bioreactors may be, e.g., submerged or suspended in liquid medium or may be attached to the surface of a solid medium. Submerged cultures may be suspended or immobilized. Suspension bioreactors can use a wider variety of organisms, since special attachment surfaces are not needed, and can operate at much larger scale than immobilized cultures. However, in a continuously operated process the organisms will be removed from the reactor with the effluent. Immobilization is a general term describing a wide variety of cell or particle attachment or entrapment. It can be applied to basically all types of biocatalysis including enzymes, cellular organelles, and cells (e.g., animal cells, plant cells, fungal cells, and bacterial cells). Immobilization is useful for continuously operated processes, since the organisms will not be removed with the reactor effluent, but is limited in scale because the cells are only present on the surfaces of the vessel. A bioreactor may also refer to a device or system meant to grow cells or tissues in the context of cell culture. The interrogation and/or monitoring of ligand levels in such samples permits the evaluation of the status of growth of the cells or production of secreted products by the cells to inform harvest or feeding or other modification of the culture. 
     Aspects of the present subject matter relate to the use of methods and biosensors provided herein to detect contamination. 
     In some embodiments, the sample comprises an environmental sample. Depending on context, there are instances in which a biological sample may also be, or may be within, an environmental sample. In certain embodiments, an environmental sample comprises a solute obtained from a biological composition, such as bone, nail, hair, shell, or cartilage. In various embodiments, an environmental sample comprises a solute obtained from an environmental substance and/or an environmental surface. For example, the solute may be dissolved/obtained from the environmental substance and/or an environmental surface using an aqueous or nonaqueous solution. In some embodiments, an aqueous may optionally comprise a nonaqueous solvent (e.g., mixed with an aqueous solvent). Non-limiting examples of environmental substances include rock, soil, clay, sand, meteorites, asteroids, dust, plastic, metal, mineral, fossils, sediment, and wood. Non-limiting examples of environmental surfaces include the surface of a vehicle such as a civilian vehicle (e.g., a satellite, a bike, a rocket, an automobile, a truck, a motorcycle, a yacht, a bus, or a plane) or a military vehicle (e.g., a tank, an armored personnel carrier, a transport truck, a jeep, a mobile artillery unit, a mobile antiaircraft unit, a minesweeper, a Mine-Resistant Ambush Protected (MRAP) vehicle, a lightweight tactical all-terrain vehicle, a high mobility multipurpose wheeled vehicle, a mobile multiple rocket launch system. an amphibious landing vehicle, a ship, a hovercraft, a submarine, a transport plane, a fighter jet, a helicopter, a rocket, or an Unmanned Arial Vehicle), a drone, a robot, a building, furniture, or an organism other than a human. In some embodiments, the sample comprises an environmental fluid. Non-limiting examples of environmental fluids include marine water, well water, drinking well water, water at the bottom of well dug for petroleum extraction or exploration, melted ice water, pond water, aquarium water, pool water, lake water, mud, stream water, river water, brook water, waste water, treated waste water, reservoir water, rain water, and ground water. In some embodiments, waste water comprises sewage water, septic tank water, agricultural runoff, water from an area in which chemical or oil spill has or is suspected of having occurred (e.g., an oil spill into a marine environment), water from an area where a radiation leak has or is suspected of having occurred (e.g., coolant from a nuclear reactor), water within the plumbing of a building, water within or exiting a research facility, and/or water within or exiting a manufacturing facility such as a factory. 
     As used herein, “suspected” with respect to an event means that there has been at least one test (e.g., a test other than a method or assay provided herein), occurrence (e.g., that is likely to or that may cause the event such as an emergency, leak, accident, flood, earthquake, storm, fire, malfunction, sunk vessel, or crash), or report (e.g., by a witness, informant, or observer) that is consistent with the event having occurred. 
     In certain embodiments, the sample comprises a food or beverage additive and/or a food or beverage composition. In some embodiments, the food or beverage composition comprises a fermented composition. In various embodiments, the sample comprises a fluid obtained from a food composition. Alternatively or in addition, the sample may comprise a solute dissolved from a food composition. In some examples, a solute is or has been dissolved from a food composition with an aqueous or nonaqueous solution. In various implementations, an aqueous solution may optionally comprise a nonaqueous solvent. In certain embodiments, a sample comprises a food composition in semisolid or liquid form. Non-limiting examples of such compositions include yogurt, soup, ice cream, a broth, a puree, a shake, a smoothie, a batter, a condiment, a sauce, and any combination thereof. In some implementations, a sample is a food engineering process (e.g., obtained from a food design, storage, transport, or production process or from equipment intended to process, transport, or store food). A food composition may comprise, e.g., a plant or a composition isolated from a plant, and/or an animal or a composition isolated from an animal. In various embodiments, a sample comprises a beverage composition. Non-limiting examples of beverage compositions include soft drinks, fountain beverages, water, coffee, tea, milk, dairy-based beverages, soy-based beverages (e.g., soy milk), almond-based beverages (e.g., almond milk), vegetable juice, fruit juice, fruit juice-flavored drinks, energy drinks, sports and fitness drinks, alcoholic products, and beverages comprising any combination thereof. Non-limiting examples of beverage compositions comprising water include purified water (e.g., filtered water, distilled water, or water purified by reverse osmosis), flavored water, mineral water, spring water, sparkling water, tonic water, and any combination thereof. In various embodiments, the sample comprises alcohol. Non-limiting examples of such samples include samples comprising or obtained/provided from beer, malt beverages, liqueur, wine, spirits, and any combination thereof. 
     In some embodiments, a sample comprises a nutritional or supplement composition. In certain implementations, the nutritional or supplement composition comprises an omega-3 fatty acid, a vitamin, a mineral, a protein powder, or a meal supplement. 
     In certain embodiments, a biosensor is implanted in a subject&#39;s body. For example, a biosensor may be implanted in a subject&#39;s blood vessel, vein, eye, natural or artificial pancreas, alimentary canal, stomach, intestine, esophagus, or skin (e.g., within the skin or under the skin). In various embodiments, the biosensor is configured within or on the surface of a contact lens. In some embodiments, the biosensor is configured to be implanted in or under the skin. In non-limiting examples, the biosensor is implanted in a subject with an optode and/or a microbead. In certain embodiments, the biosensor generates a signal transdermally. 
     The present subject matter includes a method for monitoring the level of a ligand, comprising periodically or continuously detecting the level of the ligand, wherein detecting the level of the ligand comprises (a) providing or obtaining a sample. (b) contacting the sample with a biosensor for the ligand under conditions such that the ligand-binding protein of the biosensor binds to the ligand, and (c) detecting a signal produced by the biosensor. 
     Aspects of the present subject matter also provide a method for monitoring the level of a ligand (e.g., bicarbonate or Ca 2+ ) in a subject, comprising periodically detecting the level of the ligand in the subject. Detecting the level of the ligand in the subject may comprise (a) providing or obtaining a biological sample from the subject; (b) contacting the biological sample with a biosensor for the ligand provided herein under conditions such that the ligand-binding protein of the biosensor binds to the ligand, if the ligand is present in the biological sample, and (c) detecting (i) a signal produced by a reporter group of the biosensor, or (ii) whether a signal is produced by a reporter group of the biosensor. The level of the ligand may be detected, e.g., at least once every 1, 2, 3, 6, or 12 hours, at least once every 1, 2, 3, or 4 days, at least once every 1, 2, or three weeks, or at least once every 1, 2, 3, 4, 6, or 12 months. 
     The present subject matter also provides a method for monitoring the level of a ligand in a subject. The method comprises (a) administering a biosensor provided herein or a device comprising a biosensor provided herein to the subject, wherein after administration the biosensor is in contact with a bodily fluid or surface that typically comprises the ligand, and (b) detecting (i) a signal produced by a reporter group of the biosensor continuously or repeatedly at intervals less than about 30 minutes (m), 15 m, 10 m, 5 m, 1 m, 30 seconds (s), 15 s, 10 s, 5 s, 1 s, 0.1 s, 0.001 s, 0.0001 s, or 0.00001 apart, and/or (ii) whether a signal is produced by a reporter group of the biosensor continuously or repeatedly at intervals less than about 30 m, 15 m, 10 m, 5 m, 1 m, 30 s, 15 s, 10 s, 5 s, Is, 0.1 s, 0.001 s, 0.0001 s, or 0.00001 apart. 
     Non-limiting aspects of continuously monitoring ligand levels are described in Weidemaier et al. (2011) Biosensors and Bioelectronics 26, 4117-4123 and Judge et al. (2011) Diabetes Technology &amp; Therapeutics, 13(3):309-317, the entire contents of each of which are hereby incorporated herein by reference. 
     Also within various implementations is a composition comprising a purified ligand-binding fluorescently-responsive sensor protein and a solid substrate, e.g., a particle, a bead such as a magnetic bead, or a planar surface such as a chip or slide, wherein the sensor protein is immobilized onto the solid substrate. In some embodiments, the biosensor is immobilized on a patch. In some embodiments, the patch comprises a polymer or copolymer comprising hydroxyethyl (meth)acrylate, a polyolefin, polyurethane, polystyrene, an ethyleneimethacrylic acid copolymer, an ethylene/methyl methacrylate copolymer, a polyester, and/or a polyurethane. In some embodiments, the patch comprises a woven fabric, a knitted fabric, or a nonwoven fabric of a synthetic fiber and/or natural fiber. In certain embodiments, the patch has an adhesive layer. An exemplary solid substrate solid substrate comprises a cyclic olefin copolymer. In some embodiments, the ligand-binding protein is thermostable. 
     A thermostable biosensor protein is one in which the activity (ligand binding) is retained after exposure to relatively high temperatures. For example, the urea sensor protein comprises a mid-point thermal melt transition greater than 30° C., greater than 40° C., greater than 50° C., greater than 60° C., greater than 70° C., greater than 80° C., greater than 90° C., or greater than 100° C., or about 30° C. to about 100° C., about 40° C. to about 100° C., about 50° C. to about 100° C., about 60° C. to about 100° C., about 70° C. to about 100° C., about 80° C. to about 100° C., or about 90° C. to about 100° C. In some embodiments, the sensor protein contains a single cysteine residue. In some embodiments, the single cysteine residue is located in a site of the ligand-binding protein, where it responds to ligand binding. In some examples, the protein comprises the amino acid sequence of SEQ ID NO: 64 (avBicarbBP5_18C_16F_bZif), 53 (teFeBP3_E270C), or 38 (avBicarBP5_18C), and in some examples, the single cysteine is conjugated to Badan, Acrylodan, or a derivative thereof, or Pacific Blue. For example, the derivative comprises a replacement of the two-ring naphthalene of Acrylodan or Badan with a three-ring anthracene, a fluorene, or a styrene. A reporter group is covalently bound to the single cysteine. In some situations, the solid substrate comprises a plurality of sensor proteins, each of which comprises a different dissociation constant (K d ) for ligand, e.g., for detecting and quantifying ligand levels across many ranges of concentrations. 
     The present subject matter also includes a composition comprising purified sensor protein with less than 65% identity and greater than 27% identity (e.g., 44-48% sequence identity) to any one of SEQ ID NOS: 1-28 or 75-88, wherein the sensor protein comprises a single cysteine residue, and a solid substrate, such that the sensor protein is immobilized onto the solid substrate. 
     In some embodiments, a method of detecting the presence of or the quantity of ligand in a test sample is carried out using the following steps: contacting the test sample with the biosensor or sensor protein/solid support construct to yield a complex of ligand and the ligand-binding protein or biosensor protein; contacting the complex with an excitation light; measuring an emission intensity of the reporter group from at least two wavelengths: computing a ratiometric signal from the two (or more) wavelengths; and comparing the signal to a known ligand binding curve of signals to identify the presence of or calculate the quantity of ligand in the test sample. The test sample may be obtained from a variety of sources. For example, the test sample may be selected from a bodily fluid, a food, a beverage, or a bioreactor culture broth. The testing method may be carried out in vivo, e.g., using an implantable device or dermal patch, or ex vivo. 
     In various embodiments, the subject to be tested is a mammal, e.g., a primate (such as a human, a monkey, a chimpanzee, or a gorilla), a fish, a bird, a reptile, an amphibian, or an arthropod. In some embodiments, the subject is a fish, a cow, a pig, a camel, a llama, a horse, a race horse, a work horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a wolf. a dog (e.g., a pet dog, a work dog, a police dog, or a military dog), a rat, a mouse, a seal, a whale, a manatee, a lizard, a snake, a chicken, a goose, a swan, a duck, or a penguin. 
     Exemplary Methods for Assaying the Level of Bicarbonate in Subjects 
     Aspects of the present subject matter provide a method for assaying the level of bicarbonate in a subject. The method may comprise contacting a biological sample from the subject with a biosensor for bicarbonate under conditions such that the biosensor binds to bicarbonate present in the biological sample. The biosensor comprises a reporter group attached to a bicarbonate-binding protein, and binding of bicarbonate to a bicarbonate-binding domain of the bicarbonate-binding protein causes a change in signaling by the reporter group. In various embodiments, the subject has, is suspected of having, has previously had, or is undergoing routine (e.g., during a physical) testing for a disease or an injury associated with abnormal bicarbonate levels (e.g., in a bodily fluid such as blood). Non-limiting examples of conditions and injuries that may lead to or involve high levels of bicarbonate include vomiting, dehydration, blood transfusions, overuse of medicines that contain bicarbonate (especially antacids), anorexia, chronic obstructive pulmonary disease (COPD), respiratory insufficiency, lung dysfunction, fluid in the lungs (pulmonary edema), heart disease. Cushing&#39;s disease, and Conn&#39;s syndrome. A high level of bicarbonate can be result from metabolic alkalosis, a condition that causes a pH increase in tissue. Metabolic alkalosis can happen from a loss of acid from the body, such as through vomiting and dehydration. It may also be related to conditions including anorexia and COPD. 
     As used herein, “suspected” with respect to a subject&#39;s condition (e.g., disease or injury) means that the subject has at least one symptom or test (e.g., a test other than an assay or method provided herein) that is consistent with the condition. 
     A wide range of conditions and injuries, including aspirin overdose, alcohol overdose, malnutrition, hyperthyroidism, diarrhea, kidney disease, and liver disease, uncontrolled diabetes, diabetic ketoacidosis, and a heart attack can lead to or involve low bicarbonate levels. A low level of bicarbonate may be caused by a condition called metabolic acidosis, or too much acid in the body. Metabolic acidosis is a condition that occurs when the body produces excessive quantities of acid or when the kidneys are not removing enough acid from the body. If unchecked, metabolic acidosis leads to acidemia, i.e., blood pH is low (less than 7.35) due to increased production of hydrogen ions by the body or the inability of the body to form bicarbonate in the kidney. Its causes are diverse, and its consequences can be serious, including coma and death. Together with respiratory acidosis, it is one of the two general causes of acidemia. 
     In some embodiments, the subject has a liver disease, a kidney disease (such as chronic kidney disease or an acute kidney injury), or diabetes. In some embodiments, the biological sample comprises blood, plasma, serum, sweat, tear fluid, or urine. In certain embodiments, the biological sample is present in or on the surface of the subject. In various implementations, the biosensor is applied onto or inserted into the subject. For example, the biosensor may be tattooed into the subject or is in or on a device that is implanted into the subject. In some embodiments, the biosensor may be present in or on a contact lens that is worn by the subject. Methods for determining the level of bicarbonate, e.g. in a subject who has, is suspected of having, has previously had, or is undergoing routine (e.g., during a physical) testing for a disease or an injury associated with abnormal bicarbonate levels, may be performed without other testing related to bicarbonate levels, or performed as part of a battery of clinical testing. In some embodiments, the subject has, has previously had, is suspected of having metabolic acidosis or metabolic alkalosis. 
     In some embodiments, the biological sample comprises blood, plasma, serum, sweat, tear fluid, or urine. In certain embodiments, the biological sample is present in or on the surface of the subject. In various implementations, the biosensor is applied onto or inserted into the subject. For example, the biosensor may be tattooed into the subject or is in or on a device that is implanted into the subject. In some embodiments, the biosensor may be present in or on a contact lens that is worn by the subject. Methods for determining the level of bicarbonate, e.g. in a subject who has, is suspected of having, has previously had, or is undergoing routine (e.g., during a physical) testing for a disease or an injury associated with abnormal bicarbonate levels, may be performed without other testing related to bicarbonate levels, or performed as part of a battery of clinical testing. In some embodiments, the subject is a human athlete, a soldier, a marine, a sailor, a pilot, an astronaut, a work animal (e.g., a work dog such as a sled dog. a military dog, a police dog, a rescue dog, a work horse, a police or military horse, or ox), or a performance animal (e.g., a race dog, a race camel, a race horse. a performance seal or sea lion, or a performance dolphin or porpoise). 
     Any type of abnormal bicarbonate level may be assessed, monitored or detected using the compounds, compositions, and methods provided herein. Additionally, any subject who has or is at risk of a disease or injury associated with an abnormal bicarbonate level may be assessed and/or monitored using the compounds, compositions, and methods provided herein. 
     Exemplary Methods for Assaying the Level of Ca 2+  in Subjects 
     Aspects of the present subject matter provide a method for assaying the level of a Ca 2+  in a subject. The method may comprise contacting a biological sample from the subject with a biosensor for Ca 2+  under conditions such that the biosensor binds to Ca 2  present in the biological sample. The biosensor comprises a reporter group attached to a calcium-binding protein, and binding of the Ca 2+  to a calcium-binding domain of the calcium-binding protein causes a change in signaling by the reporter group. 
     In various embodiments, the subject has, is suspected of having, has previously had, or is undergoing routine (e.g., during a physical) testing for a disease or an injury associated with abnormal Ca 2+  levels (e.g., in a bodily fluid such as blood). 
     Non-limiting examples of diseases and injuries associated with abnormal Ca 2+  levels include hypercalcemia, pancreatitis, kidney dysfunction (e.g., kidney failure, acute kidney injury or chronic kidney disease), a parathyroid disease, abnormal parathyroid gland function, hyperparathyroidism, muscle cramps, muscle spasms, muscle twitching and/or tingling in the fingers and/or around the mouth, and cancer (such as kidney, lung, or ovary cancer). Abnormally high levels of Ca 2+  may be caused by consuming too much calcium or vitamin D, hyperparathyroidism, an infection that causes granulomas such as tuberculosis and certain fungal and mycobacterial infections, multiple myeloma, T cell lymphoma and certain other cancers, metastatic bone tumors, hyperthyroidism, excessive thyroid hormone replacement drugs/treatment, Paget&#39;s disease, abnormal bone destruction and regrowth, sarcoidosis, tumors that produce a parathyroid hormone, and the administration of certain drugs such as lithium, tamoxifen, or a thiazide. 
     In some embodiments, the biological sample comprises blood, plasma, serum, sweat, tear fluid, or urine. In certain embodiments, the biological sample is present in or on the surface of the subject. In various implementations, the biosensor is applied onto or inserted into the subject. For example, the biosensor may be tattooed into the subject or is in or on a device that is implanted into the subject. In some embodiments, the biosensor may be present in or on a contact lens that is worn by the subject. Methods for determining the level of Ca 2+ , e.g. in a subject who has, is suspected of having, has previously had, or is undergoing routine (e.g., during a physical) testing for a disease or an injury associated with abnormal levels of Ca 2+ , may be performed without other testing related to Ca 2+  levels, or performed as part of a battery of clinical testing. In some embodiments, the subject is a human athlete, a soldier, a marine, a sailor, a pilot, an astronaut, a work animal (e.g., a work dog such as a sled dog, a military dog, a police dog, a rescue dog, a work horse, a police or military horse, or ox), or a performance animal (e.g., a race dog, a race camel, a race horse, a performance seal or sea lion, or a performance dolphin or porpoise). 
     Any type of abnormal Ca 2+  level may be assessed, monitored or detected using the compounds, compositions, and methods provided herein. Additionally, any subject who has or is at risk of a disease or injury associated with an abnormal cation level may be assessed and/or monitored using the compounds, compositions, and methods provided herein. 
     Exemplary Devices and Compositions Comprising Biosensors 
     Aspects of the present subject matter provide a device comprising one or more biosensors provided herein. Such devices may be, e.g., wearable, implantable, portable, or fixed. 
     In some embodiments, the device is a nanoparticle or a microparticle comprising the biosensor. Non-limiting examples of devices include devices comprising a test strip, patch, plate, bead, or chip comprising a biosensor provided herein. In certain embodiments, a device may comprise a desiccated biosensor. 
     The present subject matter also provides a contact lens or a skin patch comprising a biosensor provided herein. In some embodiments, the biosensor is throughout the contact lens or skin patch or within a particular region or zone of a contact lens or skin patch (e.g., in one or more shapes (e.g., a square, circle, or star), dots, lines, or zones, located at the periphery or a portion of the periphery of a contact lens or patch). In some embodiments, the skin patch comprises an adhesive that facilitates attachment of the patch to the surface of skin. 
     Devices provided herein may include a variety of structural compositions. For example, many polymers (including copolymers), and plastics may be used. Non-limiting examples of compositions useful in certain devices include glass, polystyrene, polypropylene, cyclic olefin copolymers, ethylene-norbornene copolymers, polyethylene, dextran, nylon, amylase, paper, a natural cellulose, a modified cellulose, a polyacrylamide, gabbros, gold, and magnetite (as well as combinations thereof). In some embodiments, the device comprises a hydrogel, a cryogel, or a soluble gel. For example, the biosensor may be incorporated into or onto the hydrogel, cryogel, or soluble gel. In various embodiments, the device comprises a matrix comprising nanopores, micropores, and/or macropores. In certain embodiments, the surface of a device comprises a polymer. In an embodiment, the surface comprises the surface of a particle or a bead having a diameter of about 0.001-1, 0.001-0.1, 0.01-0.1, 0.001-0.01, 0.1-1, 0.1-0.5, or 0.01-0.5 centimeters (cm). For example, the particle comprises a nanoparticle or a microparticle. 
     Non-limiting examples of polymers include cyclic olefin copolymers, ethylene-norbornene copolymers, polylactic acid, polyglycolic acid, agarose, alginate, poly(lactide-co-glycolide), gelatin, collagen, agarose, natural and synthetic polysaccharides, polyamino acids, poly(lysine), polyesters. polyhydroxybutyrates, polyanhydrides, polyphosphazines, polyvinyl alcohol, polyalkylene oxide, polyethylene oxide, polyallylamines, polyacrylates, modified styrene polymers, poly(4-aminomethylstyrene), pluronic polyols, poloxamers, polyuronic acid, polyvinylpyrrolidone, hydroxyethyl (meth)acrylate, polyolefins, polyurethane, polystyrene, ethylene/methacrylic acid copolymers, ethylene/methyl methacrylate copolymers, polyester, and polyurethane. In some embodiments, the patch comprises a woven fabric, a knitted fabric, or a nonwoven fabric of a synthetic fiber and/or natural fiber. 
     Non-limiting examples of temporary tattoo compositions for application to a subject&#39;s skin are discussed in U.S. Patent Application Publication No. 20090325221, published Dec. 31, 2009, and U.S. Pat. No. 6,428,797, the entire contents of each of which are incorporated herein by reference. Biosensor disclosed herein may be incorporated into any temporary tattoo or other composition for application to the skin. For example, a temporary tattoo decal for application to a subject&#39;s skin and configured to detect the presence of a ligand may comprise, e.g., a base paper or plastic: a water-soluble slip layer applied to the base paper or plastic; a temporary tattoo applied to the water-soluble release layer on the base paper, wherein the temporary tattoo comprises a biosensor disclosed herein; an adhesive layer overlying the temporary tattoo; and a protective sheet overlying the adhesive layer. 
     In some embodiments, the device comprises a plastic polymer comprising cyclic olefin copolymer (COC), such as e.g. TOPAS® COC. Several types of cyclic olefin copolymers are available based on different types of cyclic monomers and polymerization methods. Cyclic olefin copolymers are produced by chain copolymerization of cyclic monomers such as 8,9,10-trinorborn-2-ene (norbornene) or 1,2,3,4,4a,5,8,8a-octahydro-1,4:5,8-dimethanonaphthalene (tetracyclododecene) with ethene (such as TOPAS Advanced Polymer&#39;s TOPAS, Mitsui Chemical&#39;s APEL), or by ring-opening metathesis polymerization of various cyclic monomers followed by hydrogenation (Japan Synthetic Rubber&#39;s ARTON, Zeon Chemical&#39;s Zeonex and Zeonor). See, e.g., International Union of Pure and Applied Chemistry (2005)  Purr. Appl. Chem.  77(5):801-814. These later materials using a single type of monomer may be referred to as cyclic olefin polymers (COPs). A CAS Registry number for COC is 26007-43-2. 
     In some embodiments, the biosensor is covalently or noncovalently (e.g., electrostatically) attached to a surface of a device. In certain embodiments, the biosensor is attached to a surface of a device or is not attached to a surface of the device (e.g., the biosensor is physically present within the device as a component of a solution or powder but not chemically immobilized onto or into a device surface). For example, the biosensor may move within the confines of a device chamber. 
     A biosensor may be attached to a device via a variety or means, e.g., via attachment motif. In some embodiments, the attachment motif is attached to the N-terminus or the C-terminus of the biosensor. In certain embodiments, the biosensor is linked to an attachment motif via a covalent bond. In various embodiments, the biosensor is linked to the attachment motif via a linker. A non-limiting example of a linker is a polyglycine comprising 2, 3, 4, 5, or more glycines and optionally further comprising a serine. In some embodiments, the attachment motif comprises a polypeptide. Non-limiting examples of polypeptides useful in attachment moieties include hexahistidine peptides, hexalysine peptides, zinc-finger domains (ZF-QNKs), and disulfide-containing truncated zinc fingers (βZifs). An example of a hexalysine peptide comprises amino acids in the sequence of SEQ ID NO: 93, an example of a ZF-QNK comprises amino acids in the sequence of SEQ ID NO: 91, and an example of a βZif comprises amino acids in the sequence of SEQ ID NO: 90. In some embodiments, the attachment motif comprises a polypeptide that binds to plastic or cellulose. 
     The hexahistidine, hexalysine, βZif and QNK-ZF fusions enable FRSs to be immobilized onto chemically functionalized surfaces. Non-limiting aspects of chemically functionalized surfaces are discussed in Biju, V. (2014)  Chem Soc Rev,  43, 744-64 and McDonagh (2008)  Chem Rev,  108, 400-422, the entire contents of which are incorporated herein by reference. Directed evolution methods have been used to develop peptides that bind directly to non-functionalized surfaces (Care, Bergquist and Sunna 2015  Trends Biotechnol.  33, 259-68: Baneyx 2007  Curr. Opin. Biotechnol.,  18, 312-317; Gunay and Klok 2015  Bioconjug Chem,  26, 2002-15). including various plastics (Adey et al. 1995  Gene.  156, 27-31; Serizawa et al. 2005  J Am Chem Soc,  127, 13780-1; Serizawa, Sawada and Kitayama 2007a  Angew Chem Int Ed Engl.  46, 723-6; Serizawa, Sawada and Matsuno 2007b  Langmuir.  23, 11127-33: Serizawa, Techawanitchai and Matsuno 2007c  Chembiochem.  8, 989-93; Matsuno et al. 2008  Langmuir.  24, 6399-403: Chen, Serizawa and Komiyama 2011  J Pept Sci,  17, 163-8; Kumada 2010  J. Biosci. and BioEng.,  109, 583-587; Date et al. 2011  ACS Appl Mater Interfaces.  3, 351-9; Kumada 2012, Vodnik, Strukelj and Lunder 2012  J. Biotech.,  160, 222-228; Kumada 2014  Biochem, et Biophys. Acta,  1844, 1960-1969; Ejima, Matsuno and Serizawa 2010  Langmuir.  26, 17278-85), inorganic materials (Hnilova 2012  Soft Matter.  8, 4327-4334; Care et al. 2015  Trends Biotechnol.  33, 259-68), nanoparticles (Avvakumova et al. 2014  Trends Biotechnol.  32, 11-20), and cellulosic paper (Guo et al. 2013  Biomacromolecules,  14, 1795-805). Such peptides, or natural material-binding domains (Oliveira et al. 2015 Biotechnol Adv, 33, 358-69), also can be fused to FRSs to direct site-specific, oriented immobilization on their target materials while preserving FRS function. For instance, plastic-binding peptides have been developed that direct immobilization on polystyrene (Adey et al. 1995  Gene,  156, 27-31: Serizawa et al. 2007c  Chembiochem.  8, 989-93; Kumada 2010  Biochem, et Biophys. Acta.  1844, 1960-1969; Vodnik et al. 2012  Anal Biochem.  424, 83-6), polymethyl acrylate (Serizawa et al. 2005  J Am Chem Soc.  127, 13780-1; Serizawa et al. 2007a  Angew Chem Int Ed Engl,  46, 723-6; Serizawa et al. 2007b  Langmuir.  23, 11127-33; Kumada 2014  Biochem, et Biophys. Acta.  1844, 1960-1969), polycarbonate (Kumada 2012  J. Biotech.,  160, 222-228), polylactide (Matsuno et al. 2008  Langmuir,  24, 6399-403), and polyphenylene vinylene (Ejima et al. 2010  Langmuir.  26, 17278-85). Cellulose-binding peptides (Guo et al. 2013  Biomacromolecules,  14, 1795-805) and natural domains (Oliveira et al. 2015  Biotechnol Adv.  33, 358-69: Shoseyov, Shani and Levy 2006  Microbiol Mol Biol Rev.  70, 283-95) can be used to immobilize fusion proteins on paper. Inorganic material include noble metals (Hnilova 2012  Soft Matter.  8, 4327-4334), semi-conductors (Care et al. 2015  Trends Biotechnol.  33, 259-68), and fluorescent quantum dots (Medintz et al. 2005  Nat Mater.  4, 435-46; Lee et al. 2002  Science.  296, 892-5). The entire contents of each of the references above (and all other references herein) is incorporated herein by reference. 
     In some embodiments, the attachment motif is attached to a device surface and/or within a matrix of the device. In some embodiments, a biosensor is attached to an attachment motif via a covalent bond and the attachment motif is attached to a device via a covalent bond. Non-limiting examples of covalent bonds include disulfide bonds, ester bonds, thioester bonds, amide bonds, and bonds that have been formed by click reactions. Non-limiting examples of a click reaction include a reaction between an azide and an alkyne: an azide and an alkyne in the presence of Cu(I); an azide and a strained cyclooctyne: an azide and a dibenzylcyclooctyne, a difluorooctyne, or a biarylazacyclooctynone; a diaryl-strained-cyclooctyne and a 1,3-nitrone: an azide, a tetrazine, or a tetrazole and a strained alkene; an azide, a tetrazine, or a tretrazole and a oxanorbornadiene, a cyclooctene, or a trans-cycloalkene; a tetrazole and an alkene; or a tetrazole with an amino or styryl group that is activated by ultraviolet light and an alkene. 
     Alternatively or in addition, a surface of a device may be modified to contain a moiety (e.g. a reactive group) what facilitates the attachment of a biosensor and/or binds to the biosensor. In some embodiments, the biosensor is attached to a surface via a biotin-avidin interaction. 
     In various implementations, the device comprises a first region or chamber for receiving a sample and a second region or chamber that comprises the biosensor, wherein the first region or chamber is separated from the second region or chamber by a filter. In some examples, the filter is impermeable to compounds greater than about 1, 2, 3, 4, 5, 10, 50, 200, or 250 kiloDalton (kDa) in size. The sample may comprise, e.g., a tube, such as a tube that is configured for centrifugation. When sample is placed into the first region and the device is centrifuged, then a portion of the sample comprising a ligand flows through the filter into the second region where the biosensor is contacted. 
     Non-limiting examples of devices provided herein include endoscopy probes and colonoscopy probes. 
     In some embodiments, the device comprises an optode. In non-limiting examples, the optode comprises an optical fiber and a single biosensor or composite biosensor. In certain embodiments, the single biosensor or composite biosensor is immobilized on the surface or at an end of the optical fiber. In some embodiments, the optode is configured for implantation into a subject. Alternatively or in addition, the optode is configured for insertion into a sample. 
     The devices provided herein may optionally comprise a biosensor panel, a composite sensor, a sensor array, and/or a composition comprising a plurality of biosensors. In various embodiments, a device comprises multiple biosensors that detect a range of different ligand concentrations in a single sample and/or assay run (i.e., each biosensor has a different affinity for the ligand). Devices may provide spatial localization of multiple biosensors to provide the necessary addressability of different elements in a multi-sensor array comprising sensors that differ in their engineered affinities for coverage of a wide range of ligand concentrations, or sensors that each detects distinct analytes. 
     Aspects of the present subject matter provide a biosensor panel comprising a plurality of biosensors, wherein the plurality of biosensors comprises at least one biosensor disclosed herein. In some embodiments, the plurality comprises at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 biosensors. 
     The present subject matter also provides a composite sensor. The composite sensor may comprise a sensor element, wherein the sensor element comprises 2 or more biosensors, wherein at least 1 of the 2 or more biosensors is a biosensor disclosed herein. In some embodiments, the biosensors are not spatially separated in the sensor element, e.g., the biosensors are mixed within a solution, or immobilized on a surface of the sensor element. Alternatively, a mixture of different biosensors is physically present, e.g., loose, within a region or chamber of a sensor device/structure. In various embodiments, the composite sensor comprises a plurality of sensor elements, wherein each sensor element of the plurality of sensor elements comprises 2 or more biosensors, therein at least 1 of the 2 or more biosensors is a biosensor provided herein. In some embodiments, the plurality of sensor elements comprises at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 sensor elements. 
     Also included herein is a sensor array comprising a plurality of biosensors of the present subject matter. The sensor array may include, e.g., multichannel array or a multiplexed array. In some embodiments, the biosensors of the plurality of biosensors are spatially separated from each other. In certain embodiments, the biosensors are arranged linearly or in a grid on a surface of the array. 
     The present subject matter provides a composition comprising a plurality of biosensors including at least one biosensor disclosed herein. Also provided is a non-human mammal comprising a biosensor or device disclosed herein. 
     Exemplary Polypeptides and Polynucleotides 
     The present subject matter provides polynucleotides encoding any one of the polypeptides disclosed herein. The polypeptides are also provided. In various embodiments, the polynucleotides are codon-optimized for expression in a desired host cell, such as bacterial cells (e.g.,  E. coli ), yeast, insect cells, plant cells, algal cells, or mammalian cells. The polypeptides provided herein include polypeptides comprising the amino acid sequence of any one of SEQ ID NOS: 1-88. The polynucleotides provided herein include polynucleotides encoding a polypeptide comprising the amino acid sequence of any one of SEQ ID NOS: 1-88. 
     The polypeptides and biosensors provided herein may be in a variety of forms, e.g., purified in solution, dried (e.g. lyophilized) such as in the form of a powder, and in the form of a crystal (e.g., a crystal suitable for x-ray crystallography). Thus, aspects of the present subject matter provide crystal structures and crystallized forms of the ligand-binding proteins and biosensors disclosed herein. Such crystal structures and crystallized proteins are useful for designing and optimizing biosensors using principles and methods discussed herein. 
     Also provided are expression vectors comprising a polynucleotide of the present subject matter and/or encoding a polypeptide disclosed herein. Non-limiting examples of expression vectors include viral vectors and plasmid vectors. In some embodiments, an expression vector comprises nucleotides in the sequence set forth as any one of SEQ ID NOS: 95-154. In various embodiments, a polynucleotide encoding a ligand-binding protein and/or biosensor is operably linked to a promoter. The promoter may be expressed, e.g., in a prokaryotic and/or a eukaryotic cell. 
     The subject matter further includes an isolated cell comprising an expression vector provided herein. The isolated cell may be, e.g., a bacterial cell, a yeast cell, an algal cell, a plant cell, an insect cell, or a mammalian cell. Also included is a non-human multicellular organism such as a plant or an animal (e.g., an insect, a mammal, a worm, a fish, a bird, or a reptile) comprising an expression vector disclosed herein. 
     Exemplary Methods for Designing Biosensors 
     Aspects of the present subject matter provide method of identifying a candidate ligand-binding protein for use in a biosensor, comprising: (a) selecting a first protein having a known amino acid sequence (seed sequence), wherein the first protein is known to bind bicarbonate; (b) identifying a second protein having an amino acid sequence (hit sequence) with at least 15% sequence identity to the seed sequence; (c) aligning the seed amino acid sequence and the hit sequence, and comparing the hit sequence with the seed sequence at positions of the seed sequence that correspond to at least 5 primary complementary surface (PCS) amino acids, wherein each of the at least 5 PCS amino acids has a hydrogen bond interaction or a van der Waals interaction with bicarbonate when bicarbonate is bound to the first protein; and (d) identifying the second protein to be a candidate ligand-binding protein if the hit sequence comprises at least 5 amino acids that are consistent with the PCS. 
     The present subject matter also includes a method for constructing a candidate biosensor, comprising: (a) providing a candidate ligand-binding protein; (b) generating a structure of the second protein; (c) identifying at least one putative allosteric, endosteric, or peristeric site of the second protein based on the structure; (d) mutating the second protein to substitute an amino acid at the at least one putative allosteric, endosteric, or peristeric site of the second protein with a cysteine; and (e) conjugating a fluorescent compound to the cysteine. In some embodiments, the structure comprises a homology model of the second protein generated using a structure of the first protein. In some embodiments, the structure comprises a structure experimentally determined by nuclear magnetic resonance spectroscopy or X-ray crystallography. 
     Aspects of the present subject matter further provide a method for constructing a biosensor comprising a desired dissociation constant (K d ) for a ligand, comprising: (a) providing an initial biosensor that does not comprise the desired K d  for the ligand, wherein the initial biosensor is a biosensor provided herein; (b) mutating the initial biosensor to (i) alter a direct interaction in the PCS between the initial biosensor and bound ligand; (ii) manipulate the equilibrium between open and closed states of the initial biosensor: (iii) alter an interaction between the ligand-binding protein and the reporter group of the initial biosensor; or (iv) alter an indirect interaction that alters the geometry of the binding site of the biosensor, to produce a modified biosensor; and (c) selecting the modified biosensor if the modified biosensor comprises the desired K d  for the ligand. In some embodiments, the reporter group comprises Acrylodan, Badan, or a derivative thereof, and mutating the initial biosensor in (b) comprises altering an interaction between the ligand-binding protein and a carbonyl group of the Acrylodan, Badan, or derivative thereof. In some embodiments, the reporter group comprises Acrylodan, Badan, or a derivative thereof, and mutating the initial biosensor in (b) comprises altering an interaction between the ligand-binding protein and a naphthalene ring of the Acrylodan, Badan, or derivative thereof. In some embodiments, mutating the initial biosensor comprises introducing a substitution mutation into the initial biosensor. In some embodiments, the method further comprises immobilizing the affinity-tuned biosensor on a substrate. 
     In some embodiments, the second protein comprises (i) amino acids in the sequence of any one of SEQ ID NOS: 1-88: (ii) a stretch of amino acids in a sequence that is least about 95, 96, 97, 98, or 99% identical to the sequence of any one of SEQ ID NOS: 1-88: (iii) a stretch of at least about 50, 100, 150, 200, 250, 300, 350, 400, or 450 amino acids in a sequence that is at least about 95, 96, 97, 98, or 99% identical to a sequence within any one of SEQ ID NOS: 1-88; or (iv) a stretch of at least about 50, 100, 150, 200, 250, 300, 350, 400, or 450 amino acids in a sequence that is identical to a sequence within any one of SEQ ID NOS: 1-88. In various embodiments, attaching the reporter group to the putative allosteric, endosteric, or peristeric site of the first protein comprises substituting a cysteine at the site with a cysteine. For example, the reporter group is conjugated to the cysteine. Preferably, attaching a reporter group to the corresponding amino acid of the second protein produces a functional biosensor. 
     The selected first protein (e.g., the amino acid sequence thereof) may be novel or known. However, in many instances, the function of the first protein will not be known. In a non-limiting example, identifying a protein not previously known to have bicarbonate binding activity may comprise a structurally assisted functional evaluation (SAFE) homolog search method comprising the following steps: 
     (1) Collecting a sequence homology set using a BLAST sequence alignment tool starting with a bicarbonate-binding protein sequence disclosed herein or a homologue thereof as a seed. Permissive settings are used, such that pairwise hits are required to have a minimum of only, e.g., 20%, 25%, 30%, 35% or 40% sequence identity with the seed sequence. The lengths of the hit and seed are mutually constrained such that the alignment covers at least, e.g., 60%, 65%, 70%, 85%, or 90% within each partner. 
     (2) Structure-based encoding of biological function: A primary complementary surface (PCS) comprising the protein residues that form hydrogen bonds and van der Waals contacts with a bound bicarbonate is defined using computer-assisted, visual inspection of the three-dimensional structure of the protein-bicarbonate complex. This definition specifies residue positions and their permitted amino acid identity. Multiple amino acid identities are permitted at each position to encode functionally equivalent residues. This definition establishes a search filter for the accurate prediction of bicarbonate-binding proteins within the universe of sequence homologs collected in (1). For example, a candidate&#39;s residue corresponding to position 20 of synBicarbBP1 may be E: a candidate&#39;s residue corresponding to position 49 of synBicarbBP1 may be W; a candidate&#39;s residue corresponding to position 71 of synBicarbBP1 may be Q; a candidate&#39;s residue corresponding to position 102 of synBicarbBP1 may be N: a candidate&#39;s residue corresponding to position 142 of synBicarbBP1 may be T; a candidate&#39;s residue corresponding to position 148 of synBicarbBP1 may be Q; a candidate&#39;s residue corresponding to position 220 of synBicarbBP1 may be E; a candidate&#39;s residue corresponding to position 221 of synBicarbBP1 may be E. In another example, a candidate&#39;s residue corresponding to position 11 of mhFeBP1 may be R: a candidate&#39;s residue corresponding to position 102 of mhFeBP1 may be R: a candidate&#39;s residue corresponding to position 143 of mhFeBP1 may be Y; a candidate&#39;s residue corresponding to position 199 of mhFeBP1 may be Y; and a candidate&#39;s residue corresponding to position 200 of mhFeBP1 may be Y. In another example, a candidate&#39;s residue corresponding to position 11 of ttFeBP5 may be R; a candidate&#39;s residue corresponding to position 101 of ttFeBP5 may be R; a candidate&#39;s residue corresponding to position 143 of ttFeBP5 may be Y; a candidate&#39;s residue corresponding to position 200 of ttFeBP5 may be Y; and a candidate&#39;s residue corresponding to position 201 of ttFeBP5 may be Y. 
     (3) Accurate sequence alignment: Tools such as ClustalW are used to construct an accurate alignment of all the sequence homologs. The seed sequence is included in the alignment. This multiple sequence alignment establishes the equivalent positions of the seed bicarbonate-binding protein (primary complementary surface) PCS in each sequence homolog. 
     (4) Function evaluation: The bicarbonate-binding properties of each of the aligned sequence homologs is determined by measuring their compliance with the PCS sequence filter. A “Hamming distance”, H, is assigned for each homolog, which specifies the degree of sequence identity of all the residues at the aligned PCS positions. A value of H=0 indicates that the identities of all the residues at the aligned PCS positions match the amino acid(s) allowed in the PCS search filter; H&gt;0, indicates that one or more aligned positions have disallowed residues. Sequences for which H=0 are predicted to encode bicarbonate-binding proteins. 
     (5) Selection of representative SAFE homologs: The sequence homologs are ordered by (a) identity with the seed PCS, as measured by the Hamming distance. (b) fractional overall sequence identity with the seed sequence. A subset for sequences with H=0, sampling the fractional overall sequence identity is selected for experimental verification. 
     In a non-limiting example, identifying a protein not previously known to have bicarbonate-binding activity may comprise the following steps: 
     (1) performing a computational search of sequence databases to define a broad group of simple sequence or structural homologs of any known, bicarbonate-binding protein: 
     (2) using the list from step (1), deriving a search profile containing common sequence and/or structural motifs shared by the members of the list [e.g., by using computer programs such as MEME (Multiple Em for Motif Elicitation available at meme.sdsc.edu/meme/cgi-bin/meme.cgi) or BLAST]; 
     (3) searching sequence/structural databases, using a derived search profile based on the common sequence or structural motif from step (2) as query (e.g., using computer programs such as BLAST, or MAST (Motif Alignment Search Tool available at meme.sdsc.edu/meme/cgi-bin/mast.cgi), and identifying a candidate sequence, wherein a sequence homology and/or structural similarity to a reference bicarbonate-binding protein is a predetermined percentage threshold; 
     (4) compiling a list of candidate sequences to generate a list of candidate bicarbonate-binding proteins; 
     (5) expressing the candidate bicarbonate-binding proteins in a host organism; and 
     (6) testing for bicarbonate binding activity, wherein detection of bicarbonate binding in the organism (or the media thereof) indicates that the candidate sequence comprises a novel bicarbonate-binding protein. 
     In non-limiting examples, the MEME suite of sequence analysis tools (meme.sdsc.edu/meme/cgi-bin/meme.cgi) can also be used as an alternative to BLAST. Sequence motifs are discovered using the program “MEME”. These motifs can then be used to search sequence databases using the program “MAST.” The BLAST search algorithm is well-known. 
     In various embodiments relating to alignments using a ClustalW alignment program. the ClustalW alignment program may be, e.g., ClustalW alignment program version 2.1. 
     Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the invention. 
     Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
         FIG. 1A  is a cartoon and  FIGS. 1B-D  are graphs illustrating fluorescently responsive sensors.  FIG. 1A : FRSs can be constructed by site-specifically attaching a fluorophore to a protein that undergoes a conformational change upon binding ligand (triangle) in a location between the two lobes of the protein (periplasmic binding protein or engineered derivative thereof), such that the shape and intensities of the fluorescent conjugate emission spectra changes.  FIG. 1B : In the absence of ligand, the emitted fluorescence color is predominantly blue, whereas the ligand complex fluoresces green. Arrows indicate the direction of change upon ligand addition.  FIG. 1C : The ligand dependence of the absolute blue and green intensities.  FIG. 1D : The ratio of the blue and green emission intensities enables ligand binding to be determined. 
         FIG. 2  is a diagram of the bicarbonate buffering system establishes the equilibria between carbon dioxide (CO 2 ), carbonic acid (H 2 CO 3 ), hydrogen ions and bicarbonate (HCO 3   − ). This system is essential for maintaining the physiological acid-base balance. 
         FIG. 3A  shows the structure of the bicarbonate-binding protein from  Synechocyslis  PCC 6803, synBicarbBP1 [PDB identifier 2i4c (Koropatkin 2007)]. The three dimensional structure is shown. Arrows point to bicarbonate, and Ca 2+ , and residues of the primary complementary surface; small gray spheres show the location of cysteine mutations. FIG.  3 B: Ca 2+  coordination. The calcium coordination sphere forms a typical pentagonal bipyramid, with a bidentate contribution from the bound bicarbonate.  FIG. 3C : Residues involved in binding of HCO 3   −  and Ca 2+ . The PCS sequence filter used to identify the subset of bicarbonate-calcium binding proteins within a family of sequence synBicarbBP1 homologs. Residue numbering according to PDB accession 2i4c (SEQ ID NO: 166). 
         FIGS. 4A  and B show the structures of the bicarbonate-binding proteins. Arrows point to Fe III , bicarbonate, and residues of the primary complementary surface.  FIG. 4A : structure of the  Mannheimia haemolytica  bicarbonate-binding protein.  FIG. 4B : structure of the  Thermus thermophilus  bicarbonate-binding protein.  FIG. 4C : The PCS sequence filter used to identify bicarbonate binding protein. The 1SI0 seed sequence (corresponding to mhFeBP1; SEQ ID NO: 167) and the 4ELR seed sequence (corresponding to ttFeBP5; SEQ ID NO: 168) were used. 
         FIG. 5  shows an alignment of selected sequence hits using  Synechocystic  PCC 6803 as seed, with a PCS defined by its structure (PDB accession 2i4c; sequence numbering according to PDB file). Leader peptide and PCS positions indicated in gray. Ca 23 -coordinating residues underlined. 
         FIGS. 6A  and B show alignments of Fe—HCO 3  binding proteins.  FIG. 6A : Seed is  Mannheimia haemolytica  Fe—HCO 3  binding protein (sequence numbering and secondary structure assignments according to PDB accession 1si0).  FIG. 6B : Seed is  Thermus thermophilus  Fe—HCO 3  binding protein (sequence numbering and secondary structure assignments according to PDB accession 4elr). Leader peptide sequences removed in expression constructs and PCS residues are highlighted. Tyrosine residues participating in iron coordination are underlined. Note the difference in the location of the tyrosine in helix B. In the mhFeBP1 homologs it is located at the N-terminus of that helix: in the tthFeBP5 homologs it is located two residues (−½ helical turn) in from the N-terminus. 
         FIGS. 7A-C  are graphs showing the fluorescent response of avBicarbBP5 194C⋅Pacific Blue, βZif⋅Oregon Green to bicarbonate in the presence of CaCl 2  (1 mM).  FIG. 7A : Corrected emission spectra (purple line, no bicarbonate: red line, 700 mM bicarbonate: black lines, intermediate bicarbonate concentrations).  FIG. 7B : Dichromatic signal (λ 1 =455 nm, λ 2 =519 nm; black circles, experimental data points; gray lines, fit to binding isotherm,  app K d =24 mM).  FIG. 7C : Monochromatic signal (gray, 455 nm data points and fit: black, 519 nm data points and fit:  true K d =61 mM). 
         FIGS. 8A-C  are graphs showing the fluorescent response of teFeBP3 270C⋅Badan to bicarbonate.  FIG. 8A : Corrected emission spectra (purple line, no bicarbonate: red line, 700 mM bicarbonate: black lines, intermediate bicarbonate concentrations).  FIG. 8B : Dichromatic signal (λ 1 =455 nm, λ 2 =489 nm: black circles, experimental data points; gray lines, fit to binding isotherm,  app K d =48 mM).  FIG. 8C : Monochromatic signal (blue, 455 nm data points and fit: black, 489 nm data points and fit;  true K d =35 mM). 
         FIGS. 9A-C  are graphs showing the fluorescent response of avBicarbBP5 18C⋅Badan to calcium.  FIG. 9A : Corrected emission spectra Arrows indicate direction of change with increased calcium concentration  FIG. 9B : Monochromatic signal (green, 489 nm data points and fit; blue, 525 nm data points and fit;  true K d =1.0 mM).  FIG. 9C : Dichromatic signal (λ 1 =489 nm, λ 2 =525 nm: blue circles, experimental data points; red/green lines, fit to binding isotherm.  app K d =0.62 mM). 
         FIGS. 10A-C  are graphs showing that the avBicarb5 18C, 16F⋅Pacific Blue, βZif⋅5-IAF conjugate exhibits a dichromatic response to bicarbonate in the presence of CaCl 2  (1 mM).  FIG. 10A : Emission spectra. Arrows, change in intensity with increased bicarbonate concentration.  FIG. 10B : Monochromatic signal (blue, 445 nm data points and fit; green, 517 nm data points and fit;  true K d =20 mM).  FIG. 10C : Dichromatic signal (λ 1 =445 nm, λ 2 =517 nm; blue circles, experimental data points; red/green lines, fit to binding isotherm,  app K d =15 mM). 
         FIGS. 11A-C  are graphs showing that the avBicarb5 16F18C⋅Pacific Blue-βZif⋅5-IAF doubly labeled conjugate exhibits a dichromatic response to Ca 2+ .  FIG. 11A : Emission spectra. Arrows, change in intensity with increased CaCl 2  concentration.  FIG. 11B : Monochromatic signal (blue, 455 nm data points and fit; green, 520 nm data points and fit;  true K d =1.1 mM).  FIG. 11C : Dichromatic signal (λ 1 =445 nm, λ 2 =520 nm; blue circles, experimental data points: red/green lines, fit to binding isotherm,  app K d =0.96 mM). 
         FIGS. 12A-C  are diagrams showing three dominant factors that affect ngmFRET between donor and acceptors in which one partner responds to ligand binding.  FIG. 12A : Simplified Jablonski diagram illustrating radiative and non-radiative pathways in the donor and acceptor. The donor excited state (D*) is formed through illumination by the excitation source (wavy arrow) whereas the acceptor excited state (A*) is formed by resonance energy transfer (dashed arrow). The fluorescence intensity is determined by the ratio of radiative decay (gray arrows) of the excited states (gray lines) to the ground state (black line) relative to all non-radiative processes (black arrows), and the resonance energy transfer rate, k t , from donor to acceptor.  FIG. 12B : Inter-dipole geometry. Top, FRET efficiency (f=Q r /(Q 0 −Q ∞ ), where the Q r , Q 0 , Q ∞  are the quantum efficiencies at distances r, closest approach, and infinity, respectively) varies as the 6 th  power of the distance between two dipoles. Bottom, FRET efficiency varies as the square of the orientation factor κ, where κ=sin θ D  sin θ A  cos χ−2 cos θ D  cos θ A  with θ D  and θ A  the angles of the donor (blue) and acceptor (red) electronic transition dipoles with the line connecting them, and x the angle between the planes within which they lie.  FIG. 12C : Spectral overlap (gray area) between the donor fluorescence emission ( D I, blue) and acceptor fluorescence excitation (A, black) spectra. This overlap increases with bathochromic or hypsochromic shifts of the donor emission (red arrow) and acceptor excitation (dotted blue arrow) spectra, respectively. Shifts in the opposite directions decreases spectral overlap. 
         FIG. 13  shows the sequence of an exemplary synBicarbBP1 expression construct (SEQ ID NO: 95). 
         FIG. 14  shows the sequence of an exemplary teBicarbBP2 expression construct (SEQ ID NO: 96). 
         FIG. 15  shows the sequence of an exemplary ctBicarbBP3 expression construct (SEQ ID NO: 97). 
         FIG. 16  shows the sequence of an exemplary calBicarbBP4 expression construct (SEQ ID NO: 98). 
         FIG. 17  shows the sequence of an exemplary avBicarbBP5 expression construct (SEQ ID NO: 99). 
         FIG. 18  shows the sequence of an exemplary cmBicarbBP6 expression construct (SEQ ID NO: 100). 
         FIG. 19  shows the sequence of an exemplary mhFeBP1 expression construct (SEQ ID NO: 101). 
         FIG. 20  shows the sequence of an exemplary exiFeBP2 expression construct (SEQ ID NO: 102). 
         FIG. 21  shows the sequence of an exemplary teFeBP3 expression construct (SEQ ID NO: 103). 
         FIG. 22  shows the sequence of an exemplary cnFeBP4 expression construct (SEQ ID NO: 104). 
         FIG. 23  shows the sequence of an exemplary ttFeBP5 expression construct (SEQ ID NO: 105). 
         FIG. 24  shows the sequence of an exemplary msFeBP6 expression construct (SEQ ID NO: 106). 
         FIG. 25  shows the sequence of an exemplary srFeBP7 expression construct (SEQ ID NO: 107). 
         FIG. 26  shows the sequence of an exemplary hlFeBP8 expression construct (SEQ ID NO: 108). 
         FIG. 27  shows the sequence of an exemplary avBicarBP5_16C expression construct (SEQ ID NO: 109). 
         FIG. 28  shows the sequence of an exemplary avBicarBP5_17C expression construct (SEQ ID NO: 110). 
         FIG. 29  shows the sequence of an exemplary avBicarBP5_18C expression construct (SEQ ID NO: 111). 
         FIG. 30  shows the sequence of an exemplary avBicarBP5_49C expression construct (SEQ ID NO: 112). 
         FIG. 31  shows the sequence of an exemplary avBicarBP5_71C expression construct (SEQ ID NO: 113). 
         FIG. 32  shows the sequence of an exemplary avBicarBP5_140C expression construct (SEQ ID NO: 114). 
         FIG. 33  shows the sequence of an exemplary avBicarBP5_141C expression construct (SEQ ID NO: 115). 
         FIG. 34  shows the sequence of an exemplary avBicarBP5_142C expression construct (SEQ ID NO: 116). 
         FIG. 35  shows the sequence of an exemplary avBicarBP5_143C expression construct (SEQ ID NO: 117). 
         FIG. 36  shows the sequence of an exemplary avBicarBP5_146C expression construct (SEQ ID NO: 118). 
         FIG. 37  shows the sequence of an exemplary avBicarBP5_190C expression construct (SEQ ID NO: 119). 
         FIG. 38  shows the sequence of an exemplary avBicarBP5_194C expression construct (SEQ ID NO: 120). 
         FIG. 39  shows the sequence of an exemplary teFeBP3_A8C expression construct (SEQ ID NO: 121). 
         FIG. 40  shows the sequence of an exemplary teFeBP3_H10C expression construct (SEQ ID NO: 122). 
         FIG. 41  shows the sequence of an exemplary teFeBP3_D12C expression construct (SEQ ID NO: 123). 
         FIG. 42  shows the sequence of an exemplary teFeBP3_T13C expression construct (SEQ ID NO: 124). 
         FIG. 43  shows the sequence of an exemplary teFeBP3_A36C expression construct (SEQ ID NO: 125). 
         FIG. 44  shows the sequence of an exemplary teFeBP3_V58C expression construct (SEQ ID NO: 126). 
         FIG. 45  shows the sequence of an exemplary teFeBP3_R135C expression construct (SEQ ID NO: 127). 
         FIG. 46  shows the sequence of an exemplary teFeBP3_NI 39C expression construct (SEQ ID NO: 128). 
         FIG. 47  shows the sequence of an exemplary teFeBP3_I140C expression construct (SEQ ID NO: 129). 
         FIG. 48  shows the sequence of an exemplary teFeBP3_N176C expression construct (SEQ ID NO: 130). 
         FIG. 49  shows the sequence of an exemplary teFeBP3_N195C expression construct (SEQ ID NO: 131). 
         FIG. 50  shows the sequence of an exemplary teFeBP3_N268C expression construct (SEQ ID NO: 132). 
         FIG. 51  shows the sequence of an exemplary teFeBP3_E270C expression construct (SEQ ID NO: 133). 
         FIG. 52  shows the sequence of an exemplary avBicarbBP5_16C_bZif expression construct (SEQ ID NO: 134). 
         FIG. 53  shows the sequence of an exemplary avBicarbBP5_17C_bZif expression construct (SEQ ID NO: 135). 
         FIG. 54  shows the sequence of an exemplary avBicarbBP5_18C_bZif expression construct (SEQ ID NO: 136). 
         FIG. 55  shows the sequence of an exemplary avBicarbBP5_190C_bZif expression construct (SEQ ID NO: 137). 
         FIG. 56  shows the sequence of an exemplary avBicarbBP5_194C_bZif expression construct (SEQ ID NO: 138). 
         FIG. 57  shows the sequence of an exemplary avBicarbBP5_16C_71 D_bZif expression construct (SEQ ID NO: 139). 
         FIG. 58  shows the sequence of an exemplary avBicarbBP5_16C_71N_bZif expression construct (SEQ ID NO: 140). 
         FIG. 59  shows the sequence of an exemplar) avBicarbBP5_16C_71E_bZif expression construct (SEQ ID NO: 141). 
         FIG. 60  shows the sequence of an exemplary avBicarbBP5_16C_71M_bZif expression construct (SEQ ID NO: 142). 
         FIG. 61  shows the sequence of an exemplary avBicarbBP5_18C_16M_bZif expression construct (SEQ ID NO: 143). 
         FIG. 62  shows the sequence of an exemplary avBicarbBP5_18C_16F_bZif expression construct (SEQ ID NO: 144). 
         FIG. 63  shows the sequence of an exemplary avBicarbBP5_18C_16Y_bZif expression construct (SEQ ID NO: 145). 
         FIG. 64  shows the sequence of an exemplary avBicarbBP5_18C_16W_bZif expression construct (SEQ ID NO: 146). 
         FIG. 65  shows the sequence of an exemplary avBicarbBP5_18C_16E_bZif expression construct (SEQ ID NO: 147). 
         FIG. 66  shows the sequence of an exemplar) avBicarbBP5_18C_49F_bZif expression construct (SEQ ID NO: 148). 
         FIG. 67  shows the sequence of an exemplary avBicarbBP5_18C_49Y_bZif expression construct (SEQ ID NO: 149). 
         FIG. 68  shows the sequence of an exemplary avBicarbBP5_18C_141V_bZif expression construct (SEQ ID NO: 150). 
         FIG. 69  shows the sequence of an exemplary avBicarbBP5_18C_141F_bZif expression construct (SEQ ID NO: 151). 
         FIG. 70  shows the sequence of an exemplary avBicarbBP5_18C_141Y_bZif expression construct (SEQ ID NO: 152). 
         FIG. 71  shows the sequence of an exemplary avBicarbBP5_18C_141W_bZif expression construct (SEQ ID NO: 153). 
         FIG. 72  shows the sequence of an exemplary avBicarbBP5_18C_141Q_bZif expression construct (SEQ ID NO: 154). 
         FIG. 73  are illustrations of fluorophore structures. Naphthalene family (arrows indicate known or potential internal twists):  FIG. 73A  shows Acrylodan:  FIG. 73B  shows Badan:  FIG. 73C  shows IAEDANS. Xanthene family:  FIG. 73D  shows Fluorescein (5-IAF and 6-IAF);  FIG. 73E  shows Oregon Green;  FIG. 73F  shows Alexa 432:  FIG. 73G  shows Alexa532:  FIG. 73H  shows Alexa 546;  FIG. 73I  shows Texas Red. Coumarin family:  FIG. 73J  shows Pacific Blue:  FIG. 73K  shows CPM. Benzoxadiazole family:  FIG. 73L  shows IANBD. Boradiazaindacine (BODIPY) family:  FIG. 73M  shows BODIPY 499/508;  FIG. 73N  shows BODIPY 507/545. Cyanine family:  FIG. 73O  shows Cy5. Miscellaneous:  FIG. 73P  shows PyMPO. 
         FIG. 74  is a diagram relating to directly responsive partners and indirectly responsive partners in ngmFRET pathways. 
     
    
    
     DETAILED DESCRIPTION 
     Fluorescently responsive sensors (FRSs) based on engineered proteins that couple ligand-binding events to changes in the emission properties of fluorophores (being fluorescent by themselves and regardless of the presence of any other fluorophore/partner) or semi-synthetically incorporated chromophores have wide-ranging applications in cell biology and analytical chemistry. If the fluorescence emission spectrum of an engineered FRS changes shape in response to ligand binding such that the ratio of intensities at two appropriately chosen wavelengths reports on ligand concentration (dichromatic response), then ratiometric measurements can be used to monitor analyte concentrations. Ratiometry is essential for devices that rely on changes in fluorescence emission intensities, because it provides an internally consistent reference. The self-calibrating nature of a ratiometric measurement removes the necessity for carrying out on-board calibration tests prior to each measurement, obviating the need for multiple components and fluidic circuitry. Accordingly, reagentless, ratiometric fluorescent sensors have many uses in process engineering, environmental or clinical chemistry, including single-use point-of-care applications, wearable devices, or implanted “tattoos” that are interrogated transdermally. 
     The periplasmic binding protein (PBP) superfamily provide a rich source of FRSs, because PBPs combine a large diversity of ligand specificities with a common structural mechanism that is well suited to the construction of fluorescence signal transduction schemes. The three-dimensional PBP monomer structure comprises two α/β domains linked by a β-strand hinge. Binding of ligand is accompanied by a large hinge-bending motion that transitions the protein from an open to a closed state in which the ligand is enveloped within a cleft between the two domains. Semi-synthetic FRSs can be engineered with PBPs by site-specifically attaching single, thiol-reactive, environmentally sensitive fluorophores that respond to the ligand-mediated conformational change ( FIGS. 1A-D ). Semisynthetic, fluorescently labeled glucose-binding proteins in the periplasmic binding protein superfamily have been engineered successfully as reagentless, ratiometric glucose biosensors that can be used for point-of-care diagnostics and in vivo continuous glucose monitoring applications. 
     Here we present the construction of reagentless, ratiometric, fluorescent bicarbonate and calcium sensors based on engineered periplasmic binding proteins. There are two known classes of bicarbonate-binding proteins in the PBP superfamily. Both bind bicarbonate as a metal complex: either as calcium-bicarbonate, or as iron (Fe III )-bicarbonate. The Ca II —HCO 3  binding proteins typically are found in photosynthetic bacteria where they participate in HCO 3   −  uptake processes for conversion into and fixation of CO 2  in carboxysomes (Price 2008), whereas the Fe III —HCO 3  binding proteins are more wide-spread and participate in bacterial iron uptake systems (Siburt 2012). We have converted and optimized a representative member of each class into bicarbonate sensors, and optimized their performance for sensing in the clinically relevant bicarbonate concentration range (20-30 mM): the Ca II —HCO 3  binding protein from  Anabaena variabilis  (avBicarbBP5), and the Fe III —HCO 3  binding protein from  Thermosynechococcus elongatus  (teFBP3). We further engineered the Ca II —HCO 3  binding protein into a sensor for ionized calcium. Ca 2+ , with optimal performance in the 1 mM concentration range, required for sensing Ca 2+  in clinical samples. 
     Bicarbonate is an essential component for maintaining acid-base homeostasis in blood (Warrel 2010) ( FIG. 2 ). The clinical reference range for bicarbonate is 20-30 mM (Burtis 2012). Various acidosis and alkalosis conditions alter this concentration. Together with the blood pH, determination of the concentration of this important buffer therefore provides information on the pathogenesis of the imbalance (Warrel 2010). Clinical automation instrumentation uses either sample acidification in combination with a CO 2 -selective electrode, or alkalinization followed by a colorimetric coupled enzyme assay (Burtis 2012). The fluorescent sensors presented here offer a significantly simpler measurement principle. 
     There are three major forms of extracellular calcium in blood: bound to protein (albumin), complexed to phosphate or citrate, and the free Ca 2+  ion (Warrel 2010). The concentration of free Ca 2+  is tightly regulated in the 1.2-1.3 mM range (Burtis 2012). Deviations from this range are indicative of various diseases: ionized Ca 2+  determination therefore is a common clinical chemistry test (Burtis 2012). Because of the importance of Ca 2+  in intracellular biochemical signaling mechanisms, many fluorescent indicators have been developed for measuring intracellular Ca 2+  levels (Valeur 2012). These are tuned for the micromolar range and therefore are not appropriate for measuring extracellular levels. By contrast, the construction of semi-synthetic Ca 2+  biosensors based on engineered avBicarbBP5, tuned for the 0.5-10 mM range provide fluorescent sensors that are appropriate for clinical chemistry applications. 
     Biosensors 
     Biosensors are molecular recognition elements that transduce ligand-binding events into physical signals. Biosensors as detailed herein bind at least one ligand and emit a signal. A ligand-bound biosensor results in a signal that is different from the unbound biosensor. This difference facilitates detection of the at least one ligand and/or determination of ligand concentration. The biosensors may be used without the assistance of other reagents. 
     Described herein are novel engineered biosensors. These biosensors may have altered ligand-binding affinities, tailored ligand-binding specificities, and/or temperature dependencies of ligand binding or stability. For example, the herein described engineered ligand biosensors provide high-accuracy information related to extended ligand concentration ranges. 
     Binding of ligand mediates conformational changes in the biosensor, such as hinge-bending motions of the polypeptide. The conformational changes affect the environment of the reporter such that a change in the reporter-generated signal occurs. That is, without ligand bound, the biosensor results in signal generated from the reporter, and when ligand is bound, the signal generated from the reporter changes. The ligand-bound biosensor results in a reporter-generated signal that is different from the unbound biosensor. 
     In some embodiments, the methods and compositions include a plurality of a single type of biosensor. The biosensors may be identical in structure and function. For example, the biosensors of a single type may have the same polypeptide, the same reporter, and the same ligand affinity. 
     In other embodiments, the methods and compositions include a plurality of different types of biosensors. A plurality of these different types of biosensors may be arranged or incorporated in a panel. As used herein, a “panel” refers to two or more biosensors. The two or more biosensors may be different from each other. The biosensors may differ in structure and/or function. Biosensors may differ in polypeptide sequence, reporter, ligand affinities, or a combination thereof. Accordingly, there may be different types of biosensors. In some embodiments, each biosensor in the panel comprises the same reporter group. In some embodiments, each biosensor in the panel comprises a different reporter group. The panel may include at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100 biosensors. 
     The panel of biosensors includes at least one sensor element. “Sensor element” refers to a single spot, site, location, or well for the at least one biosensor, to which a sample or aliquot thereof may be applied. The panel may be a composite sensor or an array. 
     In some embodiments, the panel is a composite sensor. In a composite sensor, each sensor element includes a mixture of two or more different biosensors. In some embodiments, the composite sensor includes one sensor element. In some embodiments, the composite sensor includes two or more sensor elements. In some embodiments, signals are measured from a composite sensor in which the signals arise from one or more biosensors in the sensor element. For example, signals may be measured from a composite sensor in which the signals arise from a subset of the total number of biosensors in the sensor element. For example, signals may be measured from a composite sensor in which the signals arise from two of five biosensors in the sensor element. 
     In some embodiments, the panel is an array. In an array, each sensor element includes a single type of biosensor. An array comprises a plurality of individually and spatially localized sensor elements. Each sensor element includes a biosensor that is different than or the same as the biosensor of a different sensor element. In some embodiments, signals are measured from an array in which the signals arise separately from two or more selected biosensors in separate sensor elements. An array may comprise a plurality of sensor elements of a variety of sizes and configurations. An array may comprise a plurality of sensor elements arranged linearly. For example, an array may comprise a plurality of micrometer-sized sensor elements arranged in a single row. An array may comprise a plurality of sensor elements arranged in a grid. The grid may be two- or three-dimensional. In some embodiments, the grid is a spatially addressable grid. In some embodiments, the biosensors are incorporated into an array, such as a multichannel or multiplexed array. 
     The biosensors of the present disclosure can be used in any setting where ligand detection is required or desired, such a medical setting (e.g., determining the level of blood ligand in a subject), environmental setting (e.g., determining the level of ligand in an environmental sample), biological setting (e.g., determining the presence or amount of ligand in a reaction), or in process engineering, such as monitoring the amount of ligand in a fermentation reaction (e.g., a bacterial culture, a yeast culture, beer/wine production, etc.). Other examples include, but are not limited to, uses in the food industry (Suleiman et al, In: Biosensor Design and Application: Mathewson and Finley Eds; American Chemical Society, Washington, D.C. 1992, vol. 511); in clinical chemistry (Wilkins et al., Med. Eng. Phys. 1996, 18, 273-288: Pickup, Tr. Biotech. 1993, 11, 285-291: Meyerhoff et al., Endricon 1966, 6, 51-58; Riklin et al., Nature 1995, 376, 672-675); Willner et al., J. Am. Chem. Soc. 1996, 118, 10321-10322); as the basis for the construction of a fluorescent flow cell containing immobilized ligand binding protein-FAST conjugates (see, e.g., Wilkins et al., Med. Eng. Phys. 1966, 18, 273-288; Pickup, Tr. Biotech. 1993, 11, 285-291; Meyerhoff et al., Endricon. 1966, 6, 51; Group, New Engl. J. Med. 1993, 329, 977-986; Gough et al., Diabetes 1995, 44, 1005-1009); and in an implantable devices. 
     The biosensors as detailed herein may be administered in a variety of ways known by those of skill in the art, as appropriate for each application. Biosensors may be provided in a solution. The solution may be buffered. Biosensors may be provided in a solution and mixed directly with a sample. In some embodiments, a biosensor is immobilized onto a surface. Biosensors may be immobilized within a disposable cartridge into which a sample may be introduced or applied. Biosensors may be implanted or incorporated in a wearable device. The biosensor may be provided as an optode. 
     The biosensor may be attached to or incorporated in a wearable device. Wearable devices may include, for example, adhesive strips, patches, and contact lenses. The biosensor may be configured for placement in contact with a subject&#39;s skin or mucosal surface. In some embodiments, the biosensor is configured as an adhesive strip. In some embodiments, the biosensor is configured within or on the surface of a contact lens. In some embodiments, the contact lens is formed from a transparent substrate shaped to be worn directly over a subject&#39;s eye, as described in, for example, U.S. Pat. No. 8,608,310. 
     The biosensor may be implanted. The biosensor may be implanted in a subject&#39;s body. The biosensor may be implanted in a subject&#39;s blood vessel, vein, eye, natural or artificial pancreas, skin, or anywhere in the alimentary canal including the stomach, intestine and esophagus. The biosensor may be implanted in a subject with a microbead. In some embodiments, the biosensor is configured to be implanted in the skin. The biosensor may be implanted in a subject sub-dermally. The biosensor may generate the signal trans-dermally. In some embodiments, the biosensor may be implanted in a subject with transdermal microbeads, wherein the optical signals can be transmitted remotely between the biosensor and detecting device. 
     In some embodiments, the biosensor is administered as an optode. As used herein, “optode” refers to an optical fiber with a single biosensor, or a composite biosensor, immobilized at the surface or at the end. An “optode” may also be referred to as an “optrode.” In some embodiments, the biosensor is implanted in a subject as an optode. The optode may be incorporated with or into a needle. The optode may be incorporated with a probe such as endoscopy or colonoscopy probes. The optode may be used in a tumor, near a tumor, or at the periphery of a tumor. In some embodiments, the biosensor may be implanted in a subject as an optode, wherein the optical signals can be transmitted between the biosensor and detecting device using physical links. In some embodiments, the biosensor is administered as an optode to a sample or reaction. The optode may be contacted with a sample or reaction. In some embodiments, an optode is used to continuously or episodically monitor a ligand in a sample or reaction. 
     Methods of Detecting the Presence of a Ligand 
     Provided herein is a method of detecting the presence of a ligand in a sample. The method may include contacting the biosensor with the sample; measuring a signal from the biosensor; and comparing the signal to a ligand-free control. A difference in signal indicates the presence of ligand in the sample. 
     Also provided herein is a method of detecting the presence of ligand in a sample. The method may include (a) providing a biosensor disclosed herein in which the reporter group is attached the ligand-binding protein so that a signal transduced by the reporter group when the ligand-binding protein is bound to ligand differs from a signal transduced by the reporter group when the ligand-binding protein is not bound to ligand; (b) contacting the biosensor with the test sample under conditions such that the biosensor can bind to ligand present in the test sample; and (c) comparing the signal transduced by the reporter group when the biosensor is contacted with the test sample with the signal transduced by the reporter group when the biosensor is contacted with a ligand-free control sample, wherein a difference in the signal transduced by the reporter group when the biosensor is contacted with the test sample, as compared to when the biosensor is contacted with the control sample, indicates that the test sample contains ligand. 
     Methods of Determining the Concentration of a Ligand 
     Provided herein is a method of determining the concentration of a ligand in a sample. The method may include contacting the biosensor with the sample: measuring a signal from the biosensor; and comparing the signal to a standard hyperbolic ligand binding curve to determine the concentration of ligand in the test sample. The standard hyperbolic ligand binding curve may be prepared by measuring the signal transduced by the biosensor when contacted with control samples containing known concentrations of ligand. 
     Another aspect of the present disclosure provides a method of determining the concentration of ligand in a test sample comprising, consisting of, or consisting essentially of: (a) providing a biosensor comprising a biosensor as described herein in which the reporter group is attached the ligand-binding protein so that a signal transduced by the reporter group when the ligand-binding protein is bound to ligand differs from a signal transduced by the reporter group when the ligand-binding protein is not bound to ligand: (b) contacting the biosensor with the test sample under conditions such that the biosensor can bind to ligand present in the test sample; and (c) comparing the signal transduced by the reporter group when the biosensor is contacted with the test sample with a standard hyperbolic binding curve prepared by measuring the signal transduced by the reporter group when the biosensor is contacted with control samples containing known quantities of ligand to determine the concentration of ligand in the test sample. 
     Methods of Monitoring the Presence of a Ligand 
     The present invention is directed to a method of episodically or continuously monitoring the presence of a ligand in a reaction. In certain embodiments, the biosensors may be used in the continuous monitoring of ligand in a reaction. In certain embodiments, the ligand sensors may be used in episodic monitoring of sample aliquots. 
     The method of episodically or continuously monitoring the presence of a ligand in a reaction may include contacting the biosensor with the reaction: maintaining the reaction under conditions such that the polypeptide is capable of binding ligand present in the reaction; and episodically or continuously monitoring the signal from the biosensor in the reaction. 
     The method of episodically or continuously monitoring the presence of a ligand in a reaction may include contacting the biosensor with the reaction; maintaining the reaction under conditions such that the polypeptide is capable of binding ligand present in the reaction: episodically or continuously monitoring the signal from the biosensor in the reaction; and comparing the signal to a standard hyperbolic ligand binding curve to determine the concentration of ligand in the test sample. The standard hyperbolic ligand binding curve may be prepared by measuring the signal transduced by the biosensor when contacted with control samples containing known concentrations of ligand. 
     In some embodiments, the method further includes comparing the signal to a ligand-free control, wherein a difference in signal indicates the presence of ligand in the reaction. 
     In some embodiments, the method further includes comparing the signal to a standard hyperbolic ligand binding curve to determine the concentration of ligand in the test sample. The standard hyperbolic ligand binding curve may be prepared by measuring the signal transduced by the biosensor when contacted with control samples containing known concentrations of ligand. 
     Another aspect of the present disclosure provides a method of continuously monitoring the presence of ligand in a reaction comprising, consisting of, or consisting essentially of: (a) providing a biosensor as described herein in which the reporter group is attached the ligand-binding protein so that a signal transduced by the reporter group when the ligand-binding protein is bound to ligand differs from a signal transduced by the reporter group when the ligand-binding protein is not bound to ligand (b) maintaining the biosensor within the reaction and under conditions such that the biosensor can bind to ligand present in the reaction: (c) continuously monitoring the signal transduced by the reporter group when the biosensor is contacted with the ligand present in the reaction; and optionally (d) comparing the signal transduced by the reporter group when the biosensor is contacted with the ligand present in the reaction with the signal transduced by the reporter group when the biosensor is contacted with a ligand-free control sample, wherein a difference in the signal transduced by the reporter group when the biosensor is contacted with the ligand present in the reaction, as compared to when the biosensor is contacted with the control sample, indicates ligand is present in the reaction. 
     Yet another aspect of the present disclosure provides a method of continuously monitoring the concentration of ligand in a reaction comprising, consisting of, or consisting essentially of: (a) providing a biosensor as described herein in which the reporter group is attached the ligand-binding protein so that a signal transduced by the reporter group when the ligand-binding protein is bound to ligand differs from a signal transduced by the reporter group when the ligand-binding protein is not bound to ligand; (b) maintaining the biosensor within the reaction under conditions such that the biosensor can bind to ligand present in the reaction; and (c) continuously monitoring the signal transduced by the reporter group when the biosensor is contacted with the ligand present in the reaction; and (d) comparing the signal transduced by the reporter group when the biosensor is contacted with the ligand present in the reaction with a standard hyperbolic ligand binding curve prepared by measuring the signal transduced by the reporter group when the biosensor is contacted with control samples containing known quantities of ligand to determine the concentration of ligand in the reaction. 
     General Definitions 
     Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, and biochemistry). 
     As used herein, the term “about” in the context of a numerical value or range means±10% of the numerical value or range recited or claimed, unless the context requires a more limited range. 
     In the descriptions above and in the claims, phrases such as “at least one of” or “one or more of” may occur followed by a conjunctive list of elements or features. The term “and/or” may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it is used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases “at least one of A and B;” “one or more of A and B;” and “A and/or B” are each intended to mean “A alone, B alone, or A and B together.” A similar interpretation is also intended for lists including three or more items. For example, the phrases “at least one of A, B, and C;” “one or more of A. B, and C;” and “A, B. and/or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.” In addition, use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible 
     It is understood that where a parameter range is provided, all integers within that range, and tenths thereof, are also provided by the invention. For example, “0.2-5 mg” is a disclosure of 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg etc. up to and including 5.0 mg. 
     A small molecule is a compound that is less than 2000 daltons in mass. The molecular mass of the small molecule is preferably less than 1000 daltons, more preferably less than 600 daltons, e.g., the compound is less than 500 daltons, 400 daltons, 300 daltons, 200 daltons, or 100 daltons. 
     As used herein, an “isolated” or “purified” nucleic acid molecule, polynucleotide, polypeptide, or protein, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. Purified compounds are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%. and most preferably at least 99%, by weight the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired compound by weight. Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes/nucleic acids or sequences/amino acids that flank it in its naturally-occurring state. Purified also defines a degree of sterility that is safe for administration to a human subject, e.g., lacking infectious or toxic agents. 
     Similarly, by “substantially pure” is meant a nucleotide or polypeptide that has been separated from the components that naturally accompany it. Typically, the nucleotides and polypeptides are substantially pure when they are at least 60%, 70%, 80%, 90%, 95%, or even 99%, by weight, free from the proteins and naturally-occurring organic molecules with they are naturally associated. 
     The transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. 
     “Subject” as used herein refers to any organism from which a biological sample is obtained. For example, the sample is a biological fluid or tissue. For example, a subject is one who wants or is in need of detecting ligand or determining the concentration of ligand with the herein described biosensors. The subject may be a human or a non-human animal. The subject may be a mammal. The mammal may be a primate or a non-primate. The mammal can be a primate such as a human; a non-primate such as, for example, dog, cat, horse, cow, pig, mouse, rat, camel, llama, goat, rabbit, sheep, hamster, and guinea pig: or non-human primate such as, for example, monkey, chimpanzee, gorilla, orangutan, and gibbon. The subject may be of any age or stage of development, such as, for example, an adult, an adolescent, or an infant. 
     As used herein, an “expression vector” is a DNA or RNA vector that is capable of effecting expression of one or more polynucleotides. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically include plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in host cells of the present invention, including in one of the prokaryotic or eukaryotic cells described herein, e.g., gram-positive, gram-negative, pathogenic, non-pathogenic, commensal, cocci,  bacillus , or spiral-shaped bacterial cells; archaeal cells; or protozoan, algal, fungi, yeast, plant, animal, vertebrate, invertebrate, arthropod, mammalian, rodent, primate, or human cells. Expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the host cell and that control the expression of a polynucleotide. In particular, expression vectors of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. 
     As used herein, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a disease.” “a disease state”, or “a nucleic acid” is a reference to one or more such embodiments, and includes equivalents thereof known to those skilled in the art and so forth. 
     As used herein, “pharmaceutically acceptable” carrier or excipient refers to a carrier or excipient that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio. It can be, e.g., a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the subject. 
     The term “diagnosis” refers to a determination that a disease is present in the subject. Similarly, the term “prognosis” refers to a relative probability that a certain future outcome may occur in the subject. For example, in the context of the present disclosure, prognosis can refer to the likelihood that an individual will develop a disease, or the likely severity of the disease (e.g., severity of symptoms, rate of functional decline, survival, etc.). 
     Unless required otherwise by context, the terms “polypeptide” and “protein” are used interchangeably herein. 
     A polypeptide or class of polypeptides may be defined by the extent of identity (% identity) of its amino acid sequence to a reference amino acid sequence, or by having a greater % identity to one reference amino acid sequence than to another. A variant of any of genes or gene products disclosed herein may have, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, sequence identity to the nucleic acid or amino acid sequences described herein. The term “% identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. For example, % identity is relative to the entire length of the coding regions of the sequences being compared, or the length of a particular fragment or functional domain thereof. Variants as disclosed herein also include homologs, orthologs, or paralogs of the genes or gene products described herein. In some embodiments, variants may demonstrate a percentage of homology or identity, for example, at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 900%, 95%, 96%, 97%, 98%, or 99% identity conserved domains important for biological function, e.g., in a functional domain, e.g. a ligand-binding or catalytic domain. 
     For sequence comparison, one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Percent identity is determined using BLAST. For the BLAST searches, the following parameters were employed: (1) Expect threshold is 10; (2) Gap cost is Existence: 11 and Extension: 1; (3) The Matrix employed is BLOSUM62; (4) The filter for low complexity regions is “on.” 
     The present invention also provides for functional fragments of the genes or gene products described herein. A fragment of a protein is characterized by a length (number of amino acids) that is less than the length of the full length mature form of the protein. A fragment, in the case of these sequences and all others provided herein, may be a part of the whole that is less than the whole. Moreover, a fragment ranges in size from a single nucleotide or amino acid within a polynucleotide or polypeptide sequence to one fewer nucleotide or amino acid than the entire polynucleotide or polypeptide sequence. Finally, a fragment is defined as any portion of a complete polynucleotide or polypeptide sequence that is intermediate between the extremes defined above. 
     For example, fragments of any of the proteins or enzymes disclosed herein or encoded by any of the genes disclosed herein can be 10 to 20 amino acids, 10 to 30 amino acids, 10 to 40 amino acids, 10 to 50 amino acids, 10 to 60 amino acids, 10 to 70 amino acids, 10 to 80 amino acids, 10 to 90 amino acids, 10 to 100 amino acids, 50 to 100 amino acids, 75 to 125 amino acids, 100 to 150 amino acids, 150 to 200 amino acids, 200 to 250 amino acids, 250 to 300 amino acids, 300 to 350, 350-400, or 350-450 amino acids. The fragments encompassed in the present subject matter comprise fragments that retain functional fragments. As such, the fragments preferably retain the binding domains that are required or are important for functional activity. Fragments can be determined or generated by using the sequence information herein, and the fragments can be tested for functional activity using standard methods known in the art. For example, the encoded protein can be expressed by any recombinant technology known in the art and the binding activity of the protein can be determined. 
     As used herein a “biologically active” fragment is a portion of a polypeptide which maintains an activity of a full-length reference polypeptide. Biologically active fragments as used herein exclude the full-length polypeptide. Biologically active fragments can be any size as long as they maintain the defined activity. Preferably, the biologically active fragment maintains at least 10%, at least 50%, at least 75% or at least 90%, of the activity of the full length protein. 
     Amino acid sequence variants/mutants of the polypeptides of the defined herein can be prepared by introducing appropriate nucleotide changes into a nucleic acid defined herein, or by in vitro synthesis of the desired polypeptide. Such variants/mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final peptide product possesses the desired activity and/or specificity. 
     Mutant (altered) peptides can be prepared using any technique known in the art. For example, a polynucleotide defined herein can be subjected to in vitro mutagenesis or DNA shuffling techniques as broadly described by Harayama (1998). Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they possess, for example, ligand binding activity. 
     In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site. 
     Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues. In some embodiments, a mutated or modified protein does not comprise any deletions or insertions. In various embodiments, a mutated or modified protein has less than about 10, 9, 8, 7, 6, 5, 4, 3, or 2 deleted or inserted amino acids. 
     Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place. Sites may be substituted in a relatively conservative manner in order to maintain activity and/or specificity. Such conservative substitutions are shown in the table below under the heading of “exemplary substitutions.” 
     In certain embodiments, a mutant/variant polypeptide has only, or not more than, one or two or three or four conservative amino acid changes when compared to a naturally occurring polypeptide. Details of conservative amino acid changes are provided in the table below. As the skilled person would be aware, such minor changes can reasonably be predicted not to alter the activity of the polypeptide when expressed in a recombinant cell. 
     
       
         
           
               
               
             
               
                   
               
               
                 Original Residue 
                 Exemplary Substitutions 
               
               
                   
               
             
            
               
                 Alanine (Ala) 
                 Val; Leu; Ile; Gly 
               
               
                 Arginine (Arg) 
                 Lys 
               
               
                 Asparagine (Asn) 
                 Gln; His 
               
               
                 Cysteine (Cys) 
                 Ser 
               
               
                 Glutamine (Gln) 
                 Asn; His 
               
               
                 Glutamic Acid (Glu) 
                 Asp 
               
               
                 Glycine (Gly) 
                 Pro; Ala 
               
               
                 Histidine (His) 
                 Asn; Gln 
               
               
                 Isoleucine (Ile) 
                 Leu; Val; Ala 
               
               
                 Leucine (Leu) 
                 Ile; Val; Met; Ala; Phe 
               
               
                 Lysine (Lys) 
                 Arg 
               
               
                 Methionine (Met) 
                 Leu; Phe 
               
               
                 Phenylalanine (Phe) 
                 Leu; Val; Ala 
               
               
                 Proline (Pro) 
                 Gly 
               
               
                 Serine (Ser) 
                 Thr 
               
               
                 Threonine (Thr) 
                 Ser 
               
               
                 Tryptophan (Trp) 
                 Tyr 
               
               
                 Tyrosine (Tyr) 
                 Trp; Phe 
               
               
                 Valine (Val) 
                 Ile; Leu; Met; Phe; Ala 
               
               
                   
               
            
           
         
       
     
     Exemplary Substitutions 
     Mutations can be introduced into a nucleic acid sequence such that the encoded amino acid sequence is altered by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Certain amino acids have side chains with more than one classifiable characteristic. These families include amino acids with basic side chains (e.g., lysine. arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, tryptophan, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tyrosine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a given polypeptide is replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a given coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for given polypeptide biological activity to identify mutants that retain activity. Conversely, the invention also provides for variants with mutations that enhance or increase the endogenous biological activity. Following mutagenesis of the nucleic acid sequence, the encoded protein can be expressed by any recombinant technology known in the art and the activity/specificity of the protein can be determined. An increase, decrease, or elimination of a given biological activity of the variants disclosed herein can be readily measured by the ordinary person skilled in the art, i.e., by measuring the capability for binding a ligand and/or signal transduction. 
     In various embodiments, a polypeptide comprises mutations such that 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or less than about 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acids is substituted with a cysteine and/or a lysine. 
     Polypeptides can be produced in a variety of ways, including production and recovery of natural polypeptides or recombinant polypeptides according to methods known in the art. In one embodiment, a recombinant polypeptide is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, such as a host cell defined herein. 
     Key to the Sequence Listing 
       
     
       
         
           
               
               
             
               
                   
               
               
                 SEQ ID NO 
                 Sequence Name 
               
               
                   
               
             
            
               
                   1 
                 synBicarbBP1 [U.S. National Center for Biotechnology Information 
               
               
                   
                 (NCBI) Accession Nos. YP_005410477.1 and WP_010874027.1] 
               
               
                   
               
               
                   2 
                 teBicarbBP2 [U.S. National Center for Biotechnology Information 
               
               
                   
                 (NCB1) Accession No. NP_682790.1] 
               
               
                   
               
               
                   3 
                 ctBicarbBP3 [U.S. National Center for Biotechnology Information 
               
               
                   
                 (NCBI) Accession Nos. YP_007090308.1 and WP_015152989.1] 
               
               
                   
               
               
                   4 
                 calBicarbBP4 [U.S. National Center for Biotechnology Information 
               
               
                   
                 (NCBI) Accession Nos. YP_007137061.1 and WP_015197735.1] 
               
               
                   
               
               
                   5 
                 avBicarbBP5 [U.S. National Center for Biotechnology Information 
               
               
                   
                 (NCBI) Accession Nos. YP_321546.1 and WP_011317875.1] 
               
               
                   
               
               
                   6 
                 cmBicarbBP6 [U.S. National Center for Biotechnology Information 
               
               
                   
                 (NCBI) Accession Nos. YP_007099445.1 and WP_015162006.1] 
               
               
                   
               
               
                   7 
                 mhFeBP1 [U.S. National Center for Biotechnology Information 
               
               
                   
                 (NCBI) Accession Nos. YP_07884192.1 and WP_006253500.1] 
               
               
                   
               
               
                   8 
                 exiFeBP2 [U.S. National Center for Biotechnology Information 
               
               
                   
                 (NCBI) Accession Nos. YP_002886303.1 and WP_015880417.1] 
               
               
                   
               
               
                   9 
                 teFeBP3 [U.S. National Center for Biotechnology Information 
               
               
                   
                 (NCBI) Accession No., NP_681303.1] 
               
               
                   
               
               
                  10 
                 cnFeBP4 [U.S. National Center for Biotechnology Information 
               
               
                   
                 (NCBI) Accession Nos. YP_003796723.1 and WP_013247623.1] 
               
               
                   
               
               
                  11 
                 ttFeBP5 [U.S. National Center for Biotechnology Information 
               
               
                   
                 (NCBI) Accession No. YP_144894.1] 
               
               
                   
               
               
                  12 
                 msFeBPG [U.S. National Center for Biotechnology Information 
               
               
                   
                 (NCBI) Accession Nos. YP_003686074.1 and WP_013159102.1] 
               
               
                   
               
               
                  13 
                 srFeBP7 [U.S. National Center for Biotechnology Information 
               
               
                   
                 (NCBI) Accession Nos. YP_003572493.1 and WP_013062602.1] 
               
               
                   
               
               
                  14 
                 hlFeBP8 [U.S. National Center for Biotechnology Information 
               
               
                   
                 (NCBI) Accession Nos YP_2564837.1 and WP_012659409.1] 
               
               
                   
               
               
                  15 
                 synBicarbBP1 (with signal peptide replaced with M and a 
               
               
                   
                 GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  16 
                 teBicarbBP2 (with C247A and C260A substitution mutations, the 
               
               
                   
                 signal peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  17 
                 ctBicarbBP3 (with signal peptide replaced with M and a 
               
               
                   
                 GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  18 
                 calBicarbBP4 (with signal peptide replaced with M and 
               
               
                   
                 a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  19 
                 avBicarbBP5 (with C96A substitution mutation, signal peptide 
               
               
                   
                 replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  20 
                 cmBicarbBP6 (with C254A substitution mutation, signal peptide 
               
               
                   
                 replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  21 
                 mhFeBP1 (with C135 A and C191A substitution mutations, the 
               
               
                   
                 signal peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  22 
                 exiFeBP2 (with signal peptide replaced with M and a GGSHHHHHH 
               
               
                   
                 at C-terminus) 
               
               
                   
               
               
                  23 
                 teFeBP3 (with C184S substitution mutation, the signal peptide 
               
               
                   
                 replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  24 
                 cnFeBP4 (with signal peptide replaced with M and a GGSHHHHHH 
               
               
                   
                 at C-terminus) 
               
               
                   
               
               
                  25 
                 ttFeBPS (with signal peptide replaced with M and a GGSHHHHHH 
               
               
                   
                 at C-terminus) 
               
               
                   
               
               
                  26 
                 msFeBP6 (with signal peptide replaced with M and a GGSHHHHHH 
               
               
                   
                 at C-terminus) 
               
               
                   
               
               
                  27 
                 srFeBP7 (with signal peptide replaced with M and a GGSHHHHHH 
               
               
                   
                 at C-terminus) 
               
               
                   
               
               
                  28 
                 hlFeBP8 (with C138A and C176A substitution mutations, the signal peptide 
               
               
                   
                 replaced w ith M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  29 
                 avBicarBP5_16C (with I16C and C96A substitution mutations, 
               
               
                   
                 signal peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  30 
                 avBicarBP5_17C (with P17C and C96A substitution mutations, 
               
               
                   
                 signal peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  31 
                 avBicarBP5_18C (with I18C and C96A substitution mutations, 
               
               
                   
                 signal peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  32 
                 avBicarBP5_49C (with W49C and C96A substitution mutations, 
               
               
                   
                 signal peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  33 
                 avBicarBP5_71C (with Q71C and C96A substitution mutations, 
               
               
                   
                 signal peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  34 
                 avBicarBP5_140C (with F140C and C96A substitution mutations, 
               
               
                   
                 signal peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  35 
                 avBicarBP5_141C (with T141C and C96A substitution mutations, 
               
               
                   
                 signal peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  36 
                 avBicarBP5_142C (with F142C and C96A substitution mutations, 
               
               
                   
                 signal peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  37 
                 avBicarBP5_143C (with P143C and C96A substitution mutations, 
               
               
                   
                 signal peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  38 
                 avBicarBP5_l46C (with N146C and C96A substitution mutations, 
               
               
                   
                 signal peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  39 
                 avBicarBP5_190C (with T190C and C96A substitution mutations, 
               
               
                   
                 signal peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  40 
                 avBicarBP5_194C (with W194C and C96A substitution mutations, 
               
               
                   
                 signal peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  41 
                 teFeBP3_A8C (with A8C and C184S substitution mutations, the signal 
               
               
                   
                 peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  42 
                 teFeBP3_H10C (with H10C and C184S substitution mutations, the signal 
               
               
                   
                 peptide replaced with M, and a GGSHHHMHH at C-terminus) 
               
               
                   
               
               
                  43 
                 teFeBP3_D12C (with D12C and C184S substitution mutations, the signal 
               
               
                   
                 peptide replaced with M, and a GGSHHHMHH at C-terminus) 
               
               
                   
               
               
                  44 
                 teFeBP3_T13C (with T13C and C184S substitution mutations, the signal 
               
               
                   
                 peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  45 
                 teFeBP3_A36C (with A36C and C184S substitution mutations, the signal 
               
               
                   
                 peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  46 
                 teFeBP3_V58C (with V58C and C184S substitution mutations, the signal 
               
               
                   
                 peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  47 
                 teFeBP3_R135C (with R135C and C184S substitution mutations, the signal 
               
               
                   
                 peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  48 
                 teFeBP3_N139C (with N139C and C184S substitution mutations, the signal 
               
               
                   
                 peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  49 
                 teFeBP3_I140C (with I140C and C184S substitution mutations, the signal 
               
               
                   
                 peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  50 
                 teFeBP3_N176C (with N176C and C184S substitution mutations, the signal 
               
               
                   
                 peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  51 
                 teFeBP3_N195C (with N195C and C184S substitution mutations, the signal 
               
               
                   
                 peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  52 
                 teFeBP3_N268C (with N268C and C184S substitution mutations, the signal 
               
               
                   
                 peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  53 
                 teFeBP3_E270C (with E270C and C184S substitution mutations, the signal 
               
               
                   
                 peptide replaced with M, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  54 
                 avBicarbBP5_16C_bZif (with I16C substitution mutation, signal peptide 
               
               
                   
                 replaced with M, a bZif fusion, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  55 
                 avBicarbBP5_17C_bZif (with P17C substitution mutation, signal peptide 
               
               
                   
                 replaced with M, a bZif fusion, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  56 
                 avBicarbBP5_18C_bZif (with I18C substitution mutation, signal peptide 
               
               
                   
                 replaced with M, a bZif fusion, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  57 
                 avBicarbBP5_190C_bZif (with T190C substitution mutation, signal peptide 
               
               
                   
                 replaced with M, a bZif fusion, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  58 
                 avBicarbBP5_194C_bZif (with W194C substitution mutation, signal peptide 
               
               
                   
                 replaced with M, a bZif fusion, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  59 
                 avBicarbBP5_16C_71D_bZif (with I16C and Q71D substitution mutations, 
               
               
                   
                 signal peptide replaced with M, a bZif fusion, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  60 
                 avBicarbBP5_16C_71N_bZif (with I16C and Q71N substitution mutations, 
               
               
                   
                 signal peptide replaced with M, a bZif fusion, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  61 
                 avBicarbBP5_16C_71E_bZif (with I16C and Q71E substitution mutations, 
               
               
                   
                 signal peptide replaced with M, a bZif fusion, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  62 
                 avBicarbBP5_16C_71M_bZif (with I16C and Q71M substitution mutations, 
               
               
                   
                 signal peptide replaced with M, a bZif fusion, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  63 
                 avBicarbBP5_18C_16M_bZif (with I18C and I16M substitution mutations, 
               
               
                   
                 signal peptide replaced with M, a bZif fusion, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  64 
                 avBicarbBP5_18C_16F_bZif (with I18C and I16F substitution mutations, 
               
               
                   
                 signal peptide replaced with M, a bZif fusion, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  65 
                 avBicarbBP5_18C_16Y_bZif (with I18C and I16Y substitution mutations, 
               
               
                   
                 signal peptide replaced with M, a bZif fusion, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  66 
                 avBicarbBP5_18C_16W_bZif(with I18C and I16W substitution mutations, 
               
               
                   
                 signal peptide replaced with M, a bZif fusion, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  67 
                 avBicarbBP5_18C_16E_bZif (with I18C and I16F substitution mutations, 
               
               
                   
                 signal peptide replaced with M, a bZif fusion, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  68 
                 avBicarbBPS_18C_49F_bZif (with I18C and W49F substitution mutations, 
               
               
                   
                 signal peptide replaced with M, a bZif fusion, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  69 
                 avBicarbBP5_18C_49Y_bZif (with I18C and W49Y substitution mutations, 
               
               
                   
                 signal peptide replaced with M, a bZif fusion, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  70 
                 avBicarbBP5_18C_141V_bZif (with I18C and T141V substitution mutations, 
               
               
                   
                 signal peptide replaced with M, a bZif fusion, and a GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  71 
                 avBicarbBP5_18C_14IF_bZif (with I18C and T141F substitution 
               
               
                   
                 mutations, signal peptide replaced with M, a bZif fusion, and a 
               
               
                   
                 GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  72 
                 avBicarbBP5_18C_141Y_bZif (with I18C and T141Y substitution 
               
               
                   
                 mutations, signal peptide replaced with M, a bZif fusion, and a 
               
               
                   
                 GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  73 
                 avBicarbBP5_18C_141W_bZif (with I18C and T141W substitution 
               
               
                   
                 mutations, signal peptide replaced with M, a bZif fusion, and a 
               
               
                   
                 GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  74 
                 avBicarbBP5_18C_141Q_bZif (with I18C and T141Q substitution 
               
               
                   
                 mutations, signal peptide replaced with M, a bZif fusion, and a 
               
               
                   
                 GGSHHHHHH at C-terminus) 
               
               
                   
               
               
                  75 
                 synBicarbBP1 (with signal peptide replaced with M) 
               
               
                   
               
               
                  76 
                 eBicarbBP2 (with C247A and C260A substitution mutations and 
               
               
                   
                 signal peptide replaced with M) 
               
               
                   
               
               
                  77 
                 tBicarbBP3 (with signal peptide replaced with M) 
               
               
                   
               
               
                  78 
                 calBicarbBP4 (with signal peptide replaced with M) 
               
               
                   
               
               
                  79 
                 avBicarbBP (with C96A substitution mutation and signal peptide 
               
               
                   
                 replaced with M) 
               
               
                   
               
               
                  80 
                 cmBicarbBP6 (with C254A substitution mutation and signal peptide 
               
               
                   
                 replaced with M) 
               
               
                   
               
               
                  81 
                 mhFeBP1 (with C135A and C191A substitution mutations and 
               
               
                   
                 signal peptide replaced with M) 
               
               
                   
               
               
                  82 
                 exiFeBP2 (with signal peptide replaced with M) 
               
               
                   
               
               
                  83 
                 teFeBP3 (with C184S substitution mutation and signal peptide 
               
               
                   
                 replaced with M) 
               
               
                   
               
               
                  84 
                 cnFeBP4 (with signal peptide replaced with M) 
               
               
                   
               
               
                  85 
                 ttFeBP5 (with signal peptide replaced with M) 
               
               
                   
               
               
                  86 
                 msFeBP6 (with signal peptide replaced with M) 
               
               
                   
               
               
                  87 
                 srFeBP7 (with signal peptide replaced with M) 
               
               
                   
               
               
                  88 
                 hlFeBP8 (with C138A and C176A substitution mutations and signal 
               
               
                   
                 peptide replaced with M) 
               
               
                   
               
               
                  89 
                 GGSHHHHHH 
               
               
                   
               
               
                  90 
                 βZif 
               
               
                   
               
               
                  91 
                 ZF-QNK 
               
               
                   
               
               
                  92 
                 Hexahistidine Tag 
               
               
                   
               
               
                  93 
                 Hexalysine Tag 
               
               
                   
               
               
                  94 
                 ecGGBP (with signal peptide removed) 
               
               
                   
               
               
                  95 
                 Exemplary Expression Construct for synBicarbBP1 
               
               
                   
               
               
                  96 
                 Exemplary Expression Construct for teBicarbBP2 
               
               
                   
               
               
                  97 
                 Exemplary Expression Construct for ctBicarbBP3 
               
               
                   
               
               
                  98 
                 Exemplary Expression Construct for calBicarbBP4 
               
               
                   
               
               
                  99 
                 Exemplary Expression Construct for avBicarbBP5 
               
               
                   
               
               
                 100 
                 Exemplary Expression Construct for cmBicarbBP6 
               
               
                   
               
               
                 101 
                 Exemplary Expression Construct for mhFeBP1 
               
               
                   
               
               
                 102 
                 Exemplary Expression Construct for exiFeBP2 
               
               
                   
               
               
                 103 
                 Exemplary Expression Construct for teFeBP3 
               
               
                   
               
               
                 104 
                 Exemplary Expression Construct for cnFeBP4 
               
               
                   
               
               
                 105 
                 Exemplary Expression Construct for ttFeBP5 
               
               
                   
               
               
                 106 
                 Exemplary Expression Construct for msFeBP6 
               
               
                   
               
               
                 107 
                 Exemplary Expression Construct for srFeBP7 
               
               
                   
               
               
                 108 
                 Exemplary Expression Construct for hlFeBP8 
               
               
                   
               
               
                 109 
                 Exemplary Expression Construct for avBicarBP5_16C 
               
               
                   
               
               
                 110 
                 Exemplary Expression Construct for avBicarBP5_17C 
               
               
                   
               
               
                 111 
                 Exemplary Expression Construct for avBicarBP5_18C 
               
               
                   
               
               
                 112 
                 Exemplary Expression Construct for avBicarBP5_49C 
               
               
                   
               
               
                 113 
                 Exemplary Expression Construct for avBicarBP5_71C 
               
               
                   
               
               
                 114 
                 Exemplary Expression Construct for avBicarBP5_140C 
               
               
                   
               
               
                 115 
                 Exemplary Expression Construct for avBicarBP5_141C 
               
               
                   
               
               
                 116 
                 Exemplary Expression Construct for avBicarBP5_142C 
               
               
                   
               
               
                 117 
                 Exemplary Expression Construct for avBicarBP5_143C 
               
               
                   
               
               
                 118 
                 Exemplary Expression Construct for avBicarBP5_146C 
               
               
                   
               
               
                 119 
                 Exemplary Expression Construct for avBicarBP5_190C 
               
               
                   
               
               
                 120 
                 Exemplary Expression Construct for avBicarBP5_194C 
               
               
                   
               
               
                 121 
                 Exemplary Expression Construct for teFeBP3_A8C 
               
               
                   
               
               
                 122 
                 Exemplary Expression Construct for teFeBP3_H10C 
               
               
                   
               
               
                 123 
                 Exemplary Expression Construct for teFeBP3_D12C 
               
               
                   
               
               
                 124 
                 Exemplary Expression Construct for teFeBP3_T13C 
               
               
                   
               
               
                 125 
                 Exemplary Expression Construct for teFeBP3_A36C 
               
               
                   
               
               
                 126 
                 Exemplary Expression Construct for teFeBP3_V58C 
               
               
                   
               
               
                 127 
                 Exemplary Expression Construct for teFeBP3_R135C 
               
               
                   
               
               
                 128 
                 Exemplary Expression Construct for teFeBP3_N139C 
               
               
                   
               
               
                 129 
                 Exemplary Expression Construct for teFeBP3_I140C 
               
               
                   
               
               
                 130 
                 Exemplary Expression Construct for teFeBP3_N176C 
               
               
                   
               
               
                 131 
                 Exemplary Expression Construct for teFeBP3_N195C 
               
               
                   
               
               
                 132 
                 Exemplary Expression Construct for teFeBP3_N268C 
               
               
                   
               
               
                 133 
                 Exemplary Expression Construct for teFeBP3_E270C 
               
               
                   
               
               
                 134 
                 Exemplary Expression Construct for avBicarbBP5_16C_bZif 
               
               
                   
               
               
                 135 
                 Exemplary Expression Construct for avBicarbBP5_17C_bZif 
               
               
                   
               
               
                 136 
                 Exemplary Expression Construct for avBicarbBP5_18C bZif 
               
               
                   
               
               
                 137 
                 Exemplary Expression Construct for avBicarbBP5_190C_bZif 
               
               
                   
               
               
                 138 
                 Exemplary Expression Construct for avBicarbBP5_194C_bZif 
               
               
                   
               
               
                 139 
                 Exemplary Expression Construct for avBicarbBP5_16C_71D_bZif 
               
               
                   
               
               
                 140 
                 Exemplary Expression Construct for avBicarbBP5_16C_71N_bZif 
               
               
                   
               
               
                 141 
                 Exemplary Expression Construct for avBicarbBP5_16C_71E_bZif 
               
               
                   
               
               
                 142 
                 Exemplaiy Expression Construct for avBicarbBP5_16C_71M_bZif 
               
               
                   
               
               
                 143 
                 Exemplary Expression Construct for avBicarbBP5_18C_16M_bZif 
               
               
                   
               
               
                 144 
                 Exemplary Expression Construct for avBicarbBP5_18C_16F_bZif 
               
               
                   
               
               
                 145 
                 Exemplary Expression Construct for avBicarbBP5_18C_16Y_bZif 
               
               
                   
               
               
                 146 
                 Exemplary Expression Construct for avBicarbBP5_18C_16W_bZif 
               
               
                   
               
               
                 147 
                 Exemplary Expression Construct for avBicarbBP5_18C_16E_bZif 
               
               
                   
               
               
                 148 
                 Exemplary Expression Construct for avBicarbBP5_18C_49F_bZif 
               
               
                   
               
               
                 149 
                 Exemplary Expression Construct for avBicarbBP5_18C_49Y_bZif 
               
               
                   
               
               
                 150 
                 Exemplary Expression Construct for avBicarbBP5_18C_141V_bZif 
               
               
                   
               
               
                 151 
                 Exemplary Expression Construct for avBicarbBP5_18C_141F_bZif 
               
               
                   
               
               
                 152 
                 Exemplary Expression Construct for avBicarbBP5_18C_141Y_bZif 
               
               
                   
               
               
                 153 
                 Exemplary Expression Construct for avBicarbBP5_18C_141W_bZif 
               
               
                   
               
               
                 154 
                 Exemplary Expression Construct for avBicarbBP5_18C_141Q_bZif 
               
               
                   
               
               
                 155 
                 KLGXIXIXEXAP (conserved sequence) 
               
               
                   
               
               
                 156 
                 DGGQXQMPMP (conserved sequence) 
               
               
                   
               
               
                 157 
                 GNGIA (conserved sequence 
               
               
                   
               
               
                 158 
                 TFXXVNQD (conserved sequence 
               
               
                   
               
               
                 159 
                 HPEEY (conserved sequence) 
               
               
                   
               
               
                 160 
                 VYSXR (conserved sequence) 
               
               
                   
               
               
                 161 
                 GLXXR (conserved sequence) 
               
               
                   
               
               
                 162 
                 YYXX (conserved sequence) 
               
               
                   
               
               
                 163 
                 YXGR (conserved sequence 
               
               
                   
               
               
                 164 
                 SPAD (conserved sequence) 
               
               
                   
               
               
                 165 
                 GWXPXY (conserved sequence) 
               
               
                   
               
               
                 166 
                 2I4C seed sequence (synBicarbBP1) 
               
               
                   
               
               
                 167 
                 1SI0 seed sequence (mhFeBP1) 
               
               
                   
               
               
                 168 
                 4ELR seed sequence (ttFeBP5) 
               
               
                   
               
               
                 169 
                 ecTrx 
               
               
                   
               
               
                 170 
                 Adaptor0 
               
               
                   
               
               
                 171 
                 Adaptor1.0 
               
               
                   
               
               
                 172 
                 Adaptor2.0a 
               
               
                   
               
               
                 173 
                 Adaptor2.0b 
               
               
                   
               
               
                 174 
                 Adaptor3.0 
               
               
                   
               
               
                 175 
                 Adaptor4.0 
               
               
                   
               
               
                 176 
                 Adaptor5.0 
               
               
                   
               
               
                 177 
                 Adaptor6.0 
               
               
                   
               
               
                 178 
                 Adaptor7.0 
               
               
                   
               
               
                 179 
                 Adaptor8.0 
               
               
                   
               
               
                 180 
                 Adaptor9.0 
               
               
                   
               
               
                 181 
                 Adaptor10.0 
               
               
                   
               
               
                 182 
                 Adaptor11.0 
               
               
                   
               
               
                 183 
                 Adaptor12.0 
               
               
                   
               
               
                 184 
                 Adaptor13.0 
               
               
                   
               
               
                 185 
                 Adaptor14.0 
               
               
                   
               
               
                 186 
                 Adaptor15.0 
               
               
                   
               
               
                 187 
                 Adaptor16.0 
               
               
                   
               
            
           
         
       
     
     The terms “bZif” and “fZif” are used synonymously herein. 
     Exemplary amino acid sequences are listed below for convenience. 
     
       
         
           
               
            
               
                 synBicarbBP1 
               
               
                 SARDNVTIGSQGGGIDGGQWQMPMPHLITEGIITNGNKVPMYVLAQLITQ 
               
               
                   
               
               
                 GNGIAVAPMHEGKGVNLDITKAADYIKGFNKTNGRKFKAAHTFPNVNQDF 
               
               
                   
               
               
                 WIRYWFAAGGVDPDTDIDLLAVPPAETVQGMRNGTMDAFSTGDPWPYRIV 
               
               
                   
               
               
                 TENIGYMAGLTAQIWPYHPEEYLAIRADWVDKNPKATKALLKGIMEAQQW 
               
               
                   
               
               
                 IDDPKNRPEVVQFVSGRNYFNVPTTILESPFKGQYTMGDGQPAIDDFQKG 
               
               
                   
               
               
                 PLYWRDGIGNVSYPYRSHDLWFLTESIRWGFHRNAIPDLDTAQRIIDRVN 
               
               
                   
               
               
                 REDLWREAATEAGFTADIPSSTSRGVETFFDGITFDPANPSAYLQSLAfR 
               
               
                   
               
               
                 RVGGSHHHHHH** 
               
               
                   
               
               
                 teBicarbBP2 
               
               
                 MLETDTIRLGFIPIVESAPLIIAKERGFFARHGLTNAELSRQANWASARD 
               
               
                   
               
               
                 NVVIGSAGGGIDGGQWQMPMPYLISEGIITLNNQRLPMYVLAQLNTQGNG 
               
               
                   
               
               
                 IAISGANKGRGLHLRIADPDYIKGFAARNGRKFKAAHTFPHVNQDLWIRY 
               
               
                   
               
               
                 WFAANGIDPDRDIELLAVPPAETVAGMRNGTMDAFSTGDPWPFRIVSDDI 
               
               
                   
               
               
                 GYMATLTAQIWPYHPEEYLAVRADWVDKHPKATKALLKAVMEAQQWADDK 
               
               
                   
               
               
                 ANRPELIQIASRREYFNIPGNILTPPYEGTYTMGDGQPNFNDFNIGPLYW 
               
               
                   
               
               
                 RDPNGNSISYPYKSHDLWFLTENLRWGFNADKLKDFDNIKQMIGRVNRSD 
               
               
                   
               
               
                 LWQEAAKELGIPAAEIPTTESRGVETFFDGIKFDPDNPQAYLDSLKIKVK 
               
               
                   
               
               
                 SGGSHHHHHH** 
               
               
                   
               
               
                 ctBicarbBP3 
               
               
                 MPEQAPETTRVKLGYIPIVEAAPIIIAKEKGFFAKYGMTDVDVSKQASWG 
               
               
                   
               
               
                 SMRDNTEIGAAGGGVDGGQYQMPMPHLITEGRITKGNKPIPMYVLAQLNT 
               
               
                   
               
               
                 QGNGIAIAEKHRGKGIELELAKGGKNLFGQLKSANTPFTAAYTFAQVNQD 
               
               
                   
               
               
                 FWIRYWLAAGGVNPDADVKLIPVPAAQTVANMKTGTMDAFSTGDPWPYRI 
               
               
                   
               
               
                 VKDKIGFLAMLTADMWEFHPEEYLALRAEWVDKHPKATKALLKGIMEAQQ 
               
               
                   
               
               
                 WLDNFDNREEAAKILGGRNYFNLPAEILAGPFAGKYDMGEGRTVDDRNKA 
               
               
                   
               
               
                 VLYWRDPRGSVSYPYKSHDLWFLTESVRWGFLPPDSLTKAQALIDKVNRE 
               
               
                   
               
               
                 DLWKEAAKELGVAAADIPTSTSRGVETFFDGVKFDPENPAAYLRSLRIRR 
               
               
                   
               
               
                 AGGSHHHHHH** 
               
               
                   
               
               
                 calBicarbBP4 
               
               
                 MPEQRPETETVRLGYIPIVESAPLIIARERGLFARYGMTRVELARQASWG 
               
               
                   
               
               
                 AARDNVEIGSAGGGIDGGQWQMPMPHLITAGLITRGNREIPMYVLAQLVT 
               
               
                   
               
               
                 HGNGIAIADRHRGRGLGLRLDGARSLFRELRSSFPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAASGLDPDADVKLLTVPAAQTVANMRTGTMDAFSTGDPWPFRIVN 
               
               
                   
               
               
                 DRIGFMALLTAEMWKNHPEEYLAMRGDWVDRHPRATRAILRAVMEAQQWL 
               
               
                   
               
               
                 DNFENRREAATILAGRRYFDLSSPEILLDPYQGRYDMGDGRRIDDRLMAP 
               
               
                   
               
               
                 YYWKDERGSVSYPYRSHDLWFITENVRWGFLPRDYLANNAAKAKELrNKV 
               
               
                   
               
               
                 NREDIWREAARDLGIAAADIPTSTSRGVEEFFDGVRFDPERPEEYLRSLR 
               
               
                   
               
               
                 IRRAGVGGSHHHHHH** 
               
               
                   
               
               
                 avBicarbBP5 
               
               
                 MAEQAPEVTTVKLGYTPIVESAPLIIAKEKGFFAKYGLTNVELSRQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITRGNQRIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANRHQGRGISLRLEGARSLFSQLRSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVRLLTVPAAQTVANMRTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DRIGYMAALTAEIWRNHPEEYLAMRADWVDRYPRATRALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRREAAQILAGRNYFNLNNPEILADPYVGRYDMGDGRRIDDRSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGFKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSHHHHHH** 
               
               
                   
               
               
                 cmBicarbBP6 
               
               
                 MSSATTPETTAVKLGYIAIAESAPLIIAREKGFFARHGMTDVDVSKQASW 
               
               
                   
               
               
                 GSARDNIEIGSSNGGIDGGQWQMPMPQLISEGIITKGNRKIPMLSLAQLS 
               
               
                   
               
               
                 TQGNGIAISTQHAGKGFGLDVSGAAEYVRDMKADGKPFKAAYTFPRVNQD 
               
               
                   
               
               
                 FWIRYWLAAGGIDPNKDIDLIAVPAAQTVASMRTGSMDGFSTGDPWPSRI 
               
               
                   
               
               
                 LRDRRKYGFLAVLTAQIWPAHPEEYFAMREDWVRKHPKAAKAILKGIMEA 
               
               
                   
               
               
                 QMWADDPKNRAEMAAILAQRKYFNVPSDLLIGPYVGEYILGADRKTVKDE 
               
               
                   
               
               
                 KLAIRYWKDARGNVSYPYKSHDLWFLTESVRWGFLPQGALGEADRIINAV 
               
               
                   
               
               
                 SGEKYWREAAQELGIASADIPPSTSRGIEKFFDGAEFNPEKPKAYLDSLK 
               
               
                   
               
               
                 IKNLKAGGSHHHHHH** 
               
               
                   
               
               
                 mhFeBP1 
               
               
                 MANEVNVYSYRQPYLIEPMLKNFEKDTGIKVNIIFADKGLVDRVKQEGEL 
               
               
                   
               
               
                 SPADVLLTVDISRVMEIVNADLAQKIDSKVLEKNIPAQFRDSNDQWFGLT 
               
               
                   
               
               
                 TRARVFYTSKDRVGKLPAGFDYLDLAKPEYKGKVAVRSGKNSYNVSLFAA 
               
               
                   
               
               
                 MIEHYGlEKTKAFLEGLKANLARKPQGGDRDQVKAIKEGIADYSIGNSYY 
               
               
                   
               
               
                 YGKMLDDEKQKSWAEAAIINFPSGEHGTHKNISGVVIAKHSPNKANAVKL 
               
               
                   
               
               
                 IEYLSGEKAQGLYAELNHEYPVKEGIEPSAIVKGWGTFKSDTIKLEDIAK 
               
               
                   
               
               
                 NYEAALKLVDEVKFDDFGGSHHHHHH** 
               
               
                   
               
               
                 exiFeBP2 
               
               
                 MNVVNVYSSRHYDVDQQLYKQFEEETGIKVNVVEGKSDELLERLNTEGEN 
               
               
                   
               
               
                 TEADLFITADAGNLYQAKEAGHLQAVDSDELESNIPEKYRDTDNEWFGLT 
               
               
                   
               
               
                 KRARVIVYSKDRVKPEDLSTYEALTEEQWNGKVLVRPSENMYNISLLASF 
               
               
                   
               
               
                 IEVNGVDEAKEWAKGLVNNMARDPQGNDRDQAKAVVAGEGDVAIMNTYYM 
               
               
                   
               
               
                 GLMLNSEDEEEKKVAEQLGVFFPNQDTTGTHVNISGIAMTKASKNTENAQ 
               
               
                   
               
               
                 KLMEFMSEPSAQEKFASVNYEYPVNESVEPNELLQSWGEFKEQDINLSAL 
               
               
                   
               
               
                 GENQQEAIRIFNEVGWKGGSHHHHHH** 
               
               
                   
               
               
                 teFeBP3 
               
               
                 MVINVYSARHYDTDKALYNTFTQQTGIRVNIIEAEADALIERIRSEGSRT 
               
               
                   
               
               
                 PADVLITVDAGRLWRAQEAGILQPIQSRVLNSVVPANLREPQGHWFGLSR 
               
               
                   
               
               
                 RVRVLIYNKSRVNPSQLSTYEDLANPKWRRQILTRSSSNIYNQSLTGSLL 
               
               
                   
               
               
                 AIHGAQKTEQWARGLVQNFARPPEGNDTAQIRASAEGVGSVAIANHYYLA 
               
               
                   
               
               
                 RLIASDKEQDRAVAAKVGLFFPNQRDRGAHVNISGAGVVAGAPNRQGAIR 
               
               
                   
               
               
                 FLEYLVSPKAQEMFAMANFEYPVRAGVPVHPIVKQFGNFRGQNVNAAVFG 
               
               
                   
               
               
                 RNNAEALRIMDRAGWRGGSHHHHHH** 
               
               
                   
               
               
                 cnFeBP4 
               
               
                 MKLVVYSGRAERLIKPVLDEFQAKSGIQIELLSSGTTELVNRLQAEGDHT 
               
               
                   
               
               
                 PADVFLTNDAGSLEHARELKLLRPMNMREVERAIPSQFRAADNSWIGLSG 
               
               
                   
               
               
                 RFWIVVYNTNLVKPDQIKSLFDLTQPQWKDKIAVPNSGSEYLQAGVSVIK 
               
               
                   
               
               
                 ATFGDERTKQFLQGLKANAGTQVYQKSSQIVEAVAKGQVAAGIVNHYYIY 
               
               
                   
               
               
                 RHLATQPTAPIAAVMTDQQEGGMGAIMNVTGIGVTRASKHVESAKLLIEF 
               
               
                   
               
               
                 LVAQAGQKMFADLDKEYPLHPDVKADPTLIDRRTFRAAQVPLARLAELRE 
               
               
                   
               
               
                 ATLTLIEQVGLRGGSHHHHHH** 
               
               
                   
               
               
                 ttFeBP5 
               
               
                 MSPTLTIYSGRGQSLVEPLVKQFEAETGIRVQVRYSTDAQILAALQEEGS 
               
               
                   
               
               
                 RSPADLFWANTAGALGQASAKGLLRPLGETLLEKPIAFVPASRTWVPVTV 
               
               
                   
               
               
                 RLRVLAYNPDRIKAEELPESLLDLPRFAREKGLVGRVGWTPTYSSFQDMV 
               
               
                   
               
               
                 AGMIALYGEEKTREWLLAMKALAPKAYPSNPAMLDAIRAGEVDLGSTNHY 
               
               
                   
               
               
                 YVVRFRRAGYRLGMHHFRDGDAGNLALVTGAGLLKTSKNLAAATRFLTYL 
               
               
                   
               
               
                 LSPQAQQYFVGNIGEYPLVKGVALDPNLLPLEEALAKSPKLDLEKLPLDR 
               
               
                   
               
               
                 ALRLLRETGVLGGSHHHHHH** 
               
               
                   
               
               
                 msFeBP6 
               
               
                 MSLTLYTGRSQALVDKLVQQFQKDTGIKVNVRYGRDAEILAALQEEGSRS 
               
               
                   
               
               
                 PADVFWANTSGALEEAVKRNLLVQLPASLTRQPQEFVPSHGRWVPVSVRF 
               
               
                   
               
               
                 RVAAYNPTKVKDSDFPASVMDLPKVAKFKGRIGWTPTYSSFQDFITAMRV 
               
               
                   
               
               
                 VKGEAATKAWLQAMIAAGAKAYPSNPPMLEAMQAGEIDVALTNHYYIQRI 
               
               
                   
               
               
                 LAGVGEGEYEGKEESEEEEKKELAAREAKAGVATHYFAPGDVGGLALVTG 
               
               
                   
               
               
                 AGILATSKHQTNATRFLNYLLSKKAQPYFVDEVREYPVIAGVRVAKGMLP 
               
               
                   
               
               
                 FANAIRLSPKIDFAKLTDLEGTLKLLREVGLLGGSHHHHHH** 
               
               
                   
               
               
                 srFeBP7 
               
               
                 MLVIYSGRSKALVDSLVQQYROQADVPVRVRYGTDSQLLAALQEEGDQSP 
               
               
                   
               
               
                 ADVFWANTTGALGNAVNNGLLTELPDTLANRAARFTPSNQRWTPVTTRFR 
               
               
                   
               
               
                 VLAYNSDAVSPEDLPDSVLDLPEHEEFEGRVGWTPAYSSFQDFVTALRVT 
               
               
                   
               
               
                 EGAETARTWLSDMQALNPNSYTSNTPMVQALEAGEIDVALTNHYYVLRLK 
               
               
                   
               
               
                 HGGAEGEYEGEEEEGEEHEEEHEEEATPRASAPVEMYHFADGDLGNLALV 
               
               
                   
               
               
                 TGAGALQTSNQPDAANRFLRFLLSEQAQSFAATRVNEYPVVSGASVPDYL 
               
               
                   
               
               
                 MPADEALKMSPEFDLQKLQNMEPTLDLLRDAGALGGSHHHHHH** 
               
               
                   
               
               
                 hlFeBPX 
               
               
                 MLTVYSGRGEFLVGELVEYIEDQYDDFDLTVRYAGSTDLVNQILNEGDGS 
               
               
                   
               
               
                 PADVFYSVNAGSLGTLAGEGRSQALSSEITDMVRSEFRTEQWIGTSGRAR 
               
               
                   
               
               
                 TVPYNTGEFSDDDLPDDIMAYPEEFAGSLGWAPSYGSAQAFITAMRLIEG 
               
               
                   
               
               
                 EEATLAWLESVVEAGISSYPDEFAAAQAIADGEIDAAFTNHYYIQRVLDG 
               
               
                   
               
               
                 NPDASIGTAFTSGDAGAVFNVAGAAVVDTASDATLAENFIRHLLSAEAQD 
               
               
                   
               
               
                 YFARSTFEYPLIPDVEPIGDLPTIDELDVPDIDLTELSDLEPTIDLMREA 
               
               
                   
               
               
                 GVEVGGSHHHHHH** 
               
               
                   
               
               
                 avBicarBP5 Cysteine Scans 
               
               
                 avBicarBP5_16C 
               
               
                 MAEQAPEVTTVKLGYCPIVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSHHHHHH** 
               
               
                   
               
               
                 avBicarBP5_17C 
               
               
                 MAEQAPEVTTVKLGYICIVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSHHHHHH** 
               
               
                   
               
               
                 avBicarBP5_18C 
               
               
                 MAEQAPEVTTVKLGYIPCVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSHHHHHH** 
               
               
                   
               
               
                 avBicarBP5_49C 
               
               
                 MAEQAPEVTTVKLGYIPIVESAPLIIAKEKGFFAKYGLTNVELSKQASCG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFFFENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSHHHHHH** 
               
               
                   
               
               
                 avBicarBP5_71C 
               
               
                 MAEQAPEVTTVKLGYIPIVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWCMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSHHHHHH** 
               
               
                   
               
               
                 avBicarBP5_140C 
               
               
                 MAEQAPEVTTVKLGYIPIVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAACTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSHHHHHH** 
               
               
                   
               
               
                 avBicarBP5_141C 
               
               
                 MAEQAPEVTTVKLGYIPIVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFCFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSHHHHHH** 
               
               
                   
               
               
                 avBicarBP5_142C 
               
               
                 MAEQAPEVTTVKLGYIPIVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTCPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADVWDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSHHHHHH** 
               
               
                   
               
               
                 avBicarBP5_143C 
               
               
                 MAEQAPEVTTVKLGYIPIVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFCHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSHHHHHH** 
               
               
                   
               
               
                 avBicarBP5_146C 
               
               
                 MAEQAPEVTTVKLGYIPIVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVETGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVCQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSHHHHHH** 
               
               
                   
               
               
                 avBicarBP5_190C 
               
               
                 MAEQAPEVTTVKLGYIPIVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSCGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSHHHHHH** 
               
               
                   
               
               
                 avBicarBP5_194C 
               
               
                 MAEQAPEVTTVKLGYIPIVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPCPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSHHHHHH** 
               
               
                   
               
               
                 teFeBP3 Cysteine Scans 
               
               
                 teFeBP3_A8C 
               
               
                 MVINVYSCRHYDTDKALYNTFTQQTGIRVNIIEAEADALIERIRSEGSRT 
               
               
                   
               
               
                 PADVLITVDAGRLWRAQEAGILQPIQSRVLNSVVPANLREPQGHWFGLSR 
               
               
                   
               
               
                 RVRVLIYNKSRVNPSQLSTYEDLANPKWRRQILTRSSSNIYNQSLTGSLL 
               
               
                   
               
               
                 AIHGAQKTEQWARGLVQNFARPPEGNDTAQIRASAEGVGSVAIANHYYLA 
               
               
                   
               
               
                 RLIASDKEQDRAVAAKVGLFFPNQRDRGAHVNISGAGVVAGAPNRQGAIR 
               
               
                   
               
               
                 FLEYLVSPKAQEMFAMANFEYPVRAGVPVHPIVKQFGNFRGQNVNAAVFG 
               
               
                   
               
               
                 RNNAEALRIMDRAGWRGGSHHHHHH** 
               
               
                   
               
               
                 teFeBP3_H10C 
               
               
                 MVINVYSARCYDTDKALYNTFTQQTGIRVNIIEAEADALIERIRSEGSRT 
               
               
                   
               
               
                 PADVLITVDAGRLWRAQEAGILQPIQSRVLNSVVPANLREPQGHWFGLSR 
               
               
                   
               
               
                 RVRVLIYNKSRVNPSQLSTYEDLANPKWRRQILTRSSSNIYNQSLTGSLL 
               
               
                   
               
               
                 AIHGAQKTEQWARGLVQNFARPPEGNDTAQIRASAEGVGSVAIANHYYLA 
               
               
                   
               
               
                 RLIASDKEQDRAVAAKVGLFFPNQRDRGAHVNISGAGVVAGAPNRQGAIR 
               
               
                   
               
               
                 FLEYLVSPKAQEMFAMANFEYPVRAGVPVHPIVKQFGNFRGQNVNAAVFG 
               
               
                   
               
               
                 RNNAEALRIMDRAGWRGGSHHHHHH** 
               
               
                   
               
               
                 teFeBP3_D12C 
               
               
                 MVINVYSARHYCTDKALYNTFTQQTGIRVNIIEAEADALIERIRSEGSRT 
               
               
                   
               
               
                 PADVLITVDAGRLWRAQEAGILQPIQSRVLNSVVPANLREPQGHWFGLSR 
               
               
                   
               
               
                 RVRVLIYNKSRVNPSQLSTYEDLANPKWRRQILTRSSSNIYNQSLTGSLL 
               
               
                   
               
               
                 AIHGAQKTEQWARGLVQNFARPPEGNDTAQIRASAEGVGSVAIANHYYLA 
               
               
                   
               
               
                 RLIASDKEQDRAVAAKVGLFFPNQRDRGAHVNISGAGVVAGAPNRQGAIR 
               
               
                   
               
               
                 FLEYLVSPKAQEMFAMANFEYPVRAGVPVHPIVKQFGNFRGQNVNAAVFG 
               
               
                   
               
               
                 RNNAEALRIMDRAGWRGGSHHHHHH** 
               
               
                   
               
               
                 teFeBP3_T13C 
               
               
                 MVINVYSARHYDCDKALYNTFTQQTGIRVNIIEAEADALIERIRSEGSRT 
               
               
                   
               
               
                 PADVLITVDAGRLWRAQEAGILQPIQSRVLNSVVPANLREPQGHWFGLSR 
               
               
                   
               
               
                 RVRVLIYNKSRVNPSQLSTYEDLANPKWRRQILTRSSSNIYNQSLTGSLL 
               
               
                   
               
               
                 AIHGAQKTEQWARGLVQNFARPPEGNDTAQIRASAEGVGSVAIANHYYLA 
               
               
                   
               
               
                 RLIASDKEQDRAVAAKVGLFFPNQRDRGAHVNISGAGVVAGAPNRQGAIR 
               
               
                   
               
               
                 FLEYLVSPKAQEMFAMANFEYPVRAGVPVHPIVKQFGNFRGQNVNAAVFG 
               
               
                   
               
               
                 RNNAEALRIMDRAGWRGGSHHHHHH** 
               
               
                   
               
               
                 teFeBP3_A36C 
               
               
                 MVINVYSARHYDTDKALYNTFTQQTGIRVNIIEAECDALIERIRSEGSRT 
               
               
                   
               
               
                 PADVLITVDAGRLWRAQEAGILQPIQSRVLNSVVPANLREPQGHWFGLSR 
               
               
                   
               
               
                 RVRVLIYNKSRVNPSQLSTYEDLANPKWRRQILTRSSSNIYNQSLTGSLL 
               
               
                   
               
               
                 AIHGAQKTEQWARGLVQNFARPPEGNDTAQIRASAEGVGSVAIANHYYLA 
               
               
                   
               
               
                 RLIASDKEQDRAVAAKVGLFFPNQRDRGAHVNISGAGVVAGAPNRQGAIR 
               
               
                   
               
               
                 FLEYLVSPKAQEMFAMANFEYPVRAGVPVHPIVKQFGNFRGQNVNAAVFG 
               
               
                   
               
               
                 RNNAEALRIMDRAGWRGGSHHHHHH** 
               
               
                   
               
               
                 teFeBP3_V58C 
               
               
                 MVINVYSARHYDTDKALYNTFTQQTGIRVNIIEAEADALIERIRSEGSRT 
               
               
                   
               
               
                 PADVLITCDAGRLWRAQEAGILQPIQSRVLNSVVPANLREPQGHWFGLSR 
               
               
                   
               
               
                 RVRVLIYNKSRVNPSQLSTYEDLANPKWRRQILTRSSSNIYNQSLTGSLL 
               
               
                   
               
               
                 AIHGAQKTEQWARGLVQNFARPPEGNDTAQIRASAEGVGSVAIANHYYLA 
               
               
                   
               
               
                 RLIASDKEQDRAVAAKVGLFFPNQRDRGAHVNISGAGVVAGAPNRQGAIR 
               
               
                   
               
               
                 FLEYLVSPKAQEMFAMANFEYPVRAGVPVHPIVKQFGNFRGQNVNAAVFG 
               
               
                   
               
               
                 RNNAEALRIMDRAGWRGGSHHHHHH** 
               
               
                   
               
               
                 teFeBP3_R135C 
               
               
                 MVINVYSARHYDTDKALYNTFTQQTGIRVNIIEAEADALIERIRSEGSRT 
               
               
                   
               
               
                 PADVLITVDAGRLWRAQEAGILQPIQSRVLNSVVPANLREPQGHWFGLSR 
               
               
                   
               
               
                 RVRVLIYNKSRVNPSQLSTYEDLANPKWRRQILTCSSSNIYNQSLTGSLL 
               
               
                   
               
               
                 AIHGAQKTEQWARGLVQNFARPPEGNDTAQIRASAEGVGSVAIANHYYLA 
               
               
                   
               
               
                 RLIASDKEQDRAVAAKVGLFFPNQRDRGAHVNISGAGVVAGAPNRQGAIR 
               
               
                   
               
               
                 FEEYLVSPKAQEMFAMANFEYPVRAGVPVHPIVKQFGNFRGQNVNAAVFG 
               
               
                   
               
               
                 RNNAEALRIMDRAGWRGGSHHHHHH** 
               
               
                   
               
               
                 teFeBP3_N139C 
               
               
                 MVINVYSARHYDTDKALYNTFTQQTGIRVNIIEAEADALIERIRSEGSRT 
               
               
                   
               
               
                 PADVLITVDAGRLWRAQEAGILQPIQSRVLNSVVPANLREPQGHWFGLSR 
               
               
                   
               
               
                 RVRVLIYNKSRVNPSQLSTYEDLANPKWRRQILTRSSSCIYNQSLTGSLL 
               
               
                   
               
               
                 AIHGAQKTEQWARGLVQNFARPPEGNDTAQIRASAEGVGSVAIANHYYLA 
               
               
                   
               
               
                 RLIASDKEQDRAVAAKVGLFFPNQRDRGAHVNISGAGVVAGAPNRQGAIR 
               
               
                   
               
               
                 FLEYLVSPKAQEMFAMANFEYPVRAGVPVHPIVKQFGNFRGQNVNAAVFG 
               
               
                   
               
               
                 RNNAEALRIMDRAGWRGGSHHHHHH** 
               
               
                   
               
               
                 teFeBP3_I140C 
               
               
                 MVINVYSARHYDTDKALYNTFTQQTGIRVNIIEAEADALIERIRSEGSRT 
               
               
                   
               
               
                 PADVLITVDAGRLWRAQEAGILQPIQSRVLNSVVPANLREPQGHWFGLSR 
               
               
                   
               
               
                 RVRVLIYNKSRVNPSQLSTYEDLANPKWRRQILTRSSSNCYNQSLTGSLL 
               
               
                   
               
               
                 AIHGAQKTEQWARGLVQNFARPPEGNDTAQIRASAEGVGSVAIANHVYLA 
               
               
                   
               
               
                 RLIASDKEQDRAVAAKVGLFFPNQRDRGAHVNISGAGVVAGAPNRQGAIR 
               
               
                   
               
               
                 FLEYLVSPKAQEMFAMANFEYPVRAGVPVHPIVRQFGNFRGQNVNAAVFG 
               
               
                   
               
               
                 RNNAEALRIMDRAGWRGGSHHHHHH** 
               
               
                   
               
               
                 teFeBP3_N176C 
               
               
                 MVINVYSARHYDTDKALYNTFTQQTGIRVNIIEAEADALIERIRSEGSRT 
               
               
                   
               
               
                 PADVLITVDAGRLWRAQEAGILQPIQSRVLNSVVPANLREPQGHWFGLSR 
               
               
                   
               
               
                 RVRVLIYNKSRVNPSQLSTYEDLANPKWRRQILTRSSSNIYNQSLTGSLL 
               
               
                   
               
               
                 AIHGAQKTEQWARGLVQNFARPPEGCDTAQIRASAEGVGSVAIANHYYLA 
               
               
                   
               
               
                 RLIASDKEQDRAVAAKVGLFFPNQRDRGAHVNISGAGVVAGAPNRQGAIR 
               
               
                   
               
               
                 FLEYLVSPKAQEMFAMANFEYPVRAGVPVHPIVKQFGNFRGQNVNAAVFG 
               
               
                   
               
               
                 RNNAEALRIMDRAGWRGGSHHHHHH** 
               
               
                   
               
               
                 teFeBP3_N195C 
               
               
                 MVINVYSARHYDTDKALYNTFTQQTGIRVNIIEAEADALIERIRSEGSRT 
               
               
                   
               
               
                 PADVLITVDAGRLWRAQEAGILQPIQSRVLNSVVPANLREPQGHWFGLSR 
               
               
                   
               
               
                 RVRVLIYNKSRVNPSQLSTYEDLANPKWRRQILTRSSSNIYNQSLTGSLL 
               
               
                   
               
               
                 AIHGAQKTEQWARGLVQNFARPPEGNDTAQIRASAEGVGSVAIACHYYLA 
               
               
                   
               
               
                 RLIASDKEQDRAVAAKVGLFFPNQRDRGAHVNISGAGVVAGAPNRQGAIR 
               
               
                   
               
               
                 FLEYLVSPKAQEMFAMANFEYPVRAGVPVHPIVKQFGNFRGQNVNAAVFG 
               
               
                   
               
               
                 RNNAEALRIMDRAGWRGGSHHHHHH** 
               
               
                   
               
               
                 teFeBP3_N268C 
               
               
                 MVINVYSARHYDTDKALYNTFTQQTGIRVNIIEAEADALIERIRSEGSRT 
               
               
                   
               
               
                 PADVLITVDAGRLWRAQEAGILQPIQSRVLNSVVPANLREPQGHWFGLSR 
               
               
                   
               
               
                 RVRVLIYNKSRVNPSQLSTYEDLANPKWRRQILTRSSSNIYNQSLTGSLL 
               
               
                   
               
               
                 AIHGAQKTEQWARGLVQNFARPPEGNDTAQIRASAEGVGSVAIANHYYLA 
               
               
                   
               
               
                 RLIASDKEQDRAVAAKVGLFFPNQRDRGAHVNISGAGVVAGAPNRQGAIR 
               
               
                   
               
               
                 FLEYLVSPKAQEMFAMACFEYPVRAGVPVHPIVKQFGNFRGQNVNAAVFG 
               
               
                   
               
               
                 RNNAEALRIMDRAGWRGGSHHHHHH** 
               
               
                   
               
               
                 teFeBP3_E270C 
               
               
                 MVINVYSARHYDTDKALYNTFTQQTGIRVNIIEAEADALIERIRSEGSRT 
               
               
                   
               
               
                 PADVLITVDAGRLWRAQEAGILQPIQSRVLNSVVPANLREPQGHWFGLSR 
               
               
                   
               
               
                 RVRVLIYNKSRVNPSQLSTYEDLANPKWRRQILTRSSSNIYNQSLTGSLL 
               
               
                   
               
               
                 AIHGAQKTEQWARGLVQNFARPPEGNDTAQIRASAEGVGSVAIANHYYLA 
               
               
                   
               
               
                 RLIASDKEQDRAVAAKVGLFFPNQRDRGAHVNISGAGVVAGAPNRQGAIR 
               
               
                   
               
               
                 FLEYLVSPKAQEMFAMANFCYPVRAGVPVHPIVKQFGNFRGQNVNAAVFG 
               
               
                   
               
               
                 RNNAEALRIMDRAGWRGGSHHHHHH** 
               
               
                   
               
               
                 bZifs of Cys Scans in avBicarbP5 
               
               
                 avBicarbBP5_16C_bZif 
               
               
                 MAEQAPEVTTVKLGYCPIVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
               
                   
               
               
                 avBicarbBP5_17C_bZif 
               
               
                 MAEQAPEVTTVKLGYICIVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
               
                   
               
               
                 avBicarbBP5_18C_bZif 
               
               
                 MAEQAPEVTTVKLGYIPCVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADVWDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
               
                   
               
               
                 avBicarbBP5_190C_bZif 
               
               
                 MAEQAPEVTTVKLGYIPIVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSCGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
               
                   
               
               
                 avBicarbBP5_194C_bZif 
               
               
                 MAEQAPEVTTVKLGYIPIVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPCPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 MIEDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
               
                   
               
               
                 Affinity Variants of avBicarbP5_16C-bZif 
               
               
                 avBicarbBP5_16C_71D_bZif 
               
               
                 MAEQAPEVTTVKLGYCPIVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWDMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
               
                   
               
               
                 avBicarbBP5_116C_71N_bZif 
               
               
                 MAEQAPEVTTVKLGYCPIVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWNMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
               
                   
               
               
                 avBicarbBP5_16C_71E_bZif 
               
               
                 MAEQAPEVTTVKLGYCPIVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWEMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
               
                   
               
               
                 avBicarbBP5_16C_71M_bZif 
               
               
                 MAEQAPEVTTVKLGYCPIVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWMMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
               
                   
               
               
                 Affinity Variants of avBicarbP5_18C-bZif 
               
               
                 avBicarbBP5_18C_16M_bZif 
               
               
                 MAEQAPEVTTVKLGYMPCVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGOWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPMVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
               
                   
               
               
                 av BicarbBP5_ 18C_ 16F_bZif 
               
               
                 MAEQAPEVTTVKLGYFPCVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
               
                   
               
               
                 avBicarbBP5_18C_16Y_bZif 
               
               
                 MAEQAPEVTTVkLGYYPCVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
               
                   
               
               
                 avBicarbBP5_18C_16W_bZif 
               
               
                 MAEQAPEVTTVKLGYWPCVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWOMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
               
                   
               
               
                 avBicarbBP5_18C_16E_bZif 
               
               
                 MAEQAPEVTTVKLGYEPCVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
               
                   
               
               
                 avBicarbBP5_18C_49F_bZif 
               
               
                 MAEQAPEVTTVKLGYIPCVESAPLIIAKEKGFFAKYGLTNVELSKQASFG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSNHHHHFI** 
               
               
                   
               
               
                 avBicarbBP5_18C_49Y_bZif 
               
               
                 MAEQAPEVTTVKLGYIPCVESAPLIIAKEKGFFAKYGLTNVELSKQASYG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFTFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
               
                   
               
               
                 avBicarbBP5_18C_141V_bZif 
               
               
                 MAEQAPEVTTVKLGYIPCVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGRGISLKLEGAKSLFSQLKSSTPFTAAFVFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
               
                   
               
               
                 avBicarbBP5_18C_141F_bZif 
               
               
                 MAEQAPEVTTVKLGYIPCVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFFFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
               
                   
               
               
                 avBicarbBP5_18C_141Y_bZif 
               
               
                 MAEQAPEVTTVKLGYIPCVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFYFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
               
                   
               
               
                 avBicarbBP5_18C_141W_bZif 
               
               
                 MAEQAPEVTTVKLGYIPCVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFWFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAREAGIAAADIPTSISRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
               
                   
               
               
                 avBicarbBP5_18C_141Q_bZif 
               
               
                 MAEQAPEVTTVKLGYIPCVESAPLIIAKEKGFFAKYGLTNVELSKQASWG 
               
               
                   
               
               
                 SARDNVEIGSAGGGIDGGQWQMPMPHLITEGLITKGNQKIPMYVLAQLIT 
               
               
                   
               
               
                 HGNGIAIANKHQGKGISLKLEGAKSLFSQLKSSTPFTAAFQFPHVNQDLW 
               
               
                   
               
               
                 IRYWLAAGGIDPDADVKLLTVPAAQTVANMKTGTMDAFSTGDPWPFRLVN 
               
               
                   
               
               
                 DKIGYMAALTAEIWKNHPEEYLAMRADWVDKYPKATKALLKGIMEAQQWL 
               
               
                   
               
               
                 DNFDNRKEAAQILAGRNYFNLNNPEILADPYVGKYDMGDGRKIDDKSMAA 
               
               
                   
               
               
                 YYWKDEKGSVSYPYKSHDLWFITENVRWGFLPKDYLANGAAKAKELIDKV 
               
               
                   
               
               
                 NREDIWKEAAKEAGIAAADIPTSTSRGVEEFFDGTKFDPEKPDEYLKSLK 
               
               
                   
               
               
                 IKKVSVGGSTGEKPYKCPECGKSFSRSGGSHHHHHH** 
               
            
           
         
       
     
     Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results. 
     Example 1. Fluorescently Responsive Sensor Engineering Phases 
     The engineering of FRSs can be divided into five phases:
         1. Binding protein discovery. A set of binding protein sequence homologs is identified for the target ligand. Accurate assignment of their ligand-binding function requires application of a prediction method that incorporates information encoded in the experimentally determined three-dimensional structure of known periplasmic binding proteins for the target ligand.   2. Experimental lead validation. Synthetic genes are constructed, which are optimized for heterologous expression in  Escherichia coli  of one or more predicted binding protein sequences for the target ligand. The ligand binding properties and thermostabilities of the corresponding expressed, purified proteins are evaluated.   3. Engineering of fluorescent responses. Semisynthetic fluorescent conjugates of the experimentally validated leads are constructed by first attaching single fluorophores to single cysteine mutants. The effect of ligand binding on the fluorescence emission properties of those conjugates is evaluated. The spectral properties of a subset of responsive fluorophores is improved using a double-labeling strategy in which a second fluorophore is site-specifically attached to a small domain fused to the N- or C-terminus to establish ligand-modulated fluorescence resonance energy transfer. Those singly or doubly labeled conjugates that evince strong, ratiometric responses are selected as FRSs for use in sensing applications.   4. Affinity tuning. Single or multiple mutations are introduced by site-directed mutagenesis to alter the ligand-binding affinities of ligand-responsive FRSs. A set of FRS variants is selected that together cover the clinical ligand concentration range with high accuracy.       

     Example 2. Sensor Engineering Phase 1: Identification of a Family of Periplasmic Binding Proteins Homologs for the Target Ligand Using Structurally Assisted Function Evaluation 
     As a first step in constructing robust sensor candidates for the target ligand, we examined bacterial genomic sequences to identify periplasmic binding protein sequences for bicarbonate and calcium in known thermophiles. Homologs from such organisms are likely to encode thermostable proteins (Urbieta et al. 2015). Analysis of enzyme families has shown that overall sequence identity below ˜60% is a weak predictor of function conservation (Todd 2001, Tian 2003). Furthermore, functional assignments based on sequence homology alone are known to be particularly problematic in the PBP superfamily. For instance. PBPs that by overall sequence identity are predicted to bind oligopeptides were found to bind oligosaccharides (Cuneo, Beese and Hellinga 2009, Nanavati 2006). Enzyme functional assignments are improved greatly if a sequence selection filter based on conservation of catalytic residues identified from protein structures is included (George 2005). Such catalytic residues comprise a subset of all the residues that contact an enzyme substrate or inhibitor (George 2005). In the case of the PBPs, functional selection filters need to take into account all the protein-ligand contacts that encode the ligand-binding function. Accordingly, we have developed a structurally assisted functional evaluation (SAFE) method to identify PBP sequence homologs with accurately predicted function. The SAFE homolog search method consists of five steps:
         1. Sequence homolog set is collected using the BLAST sequence alignment tool (Altschul et al. 1990), starting with a seed sequence. The following BLAST parameters: (1) Expect threshold is 10.0; (2) Gap cost is Existence: 11 and Extension: 1: (3) The Matrix employed is BLOSUM62; (4) The filter for low complexity regions is “on.” Permissive settings are used, such that pairwise hits are required to have a minimum of only 20% sequence identity with the seed sequence. The lengths of the hit and seed are mutually constrained such that the alignment covers at least 70% within each partner. This set of sequences defines a universe of possible ligand-binding proteins for the target ligand without accurately assigning function.   2. Structure-based encoding of biological function. A primary complementary surface comprising the protein residues that form hydrogen bonds and van der Waals contacts with the bound complex is defined using computer-assisted, visual inspection of the three-dimensional structure of the ligand bound complex. This definition specifies residue positions and their permitted amino acid identity. Multiple amino acid identities are permitted at each position to encode functionally equivalent residues. This definition establishes a search filter for the accurate prediction of binding proteins for the target ligand within the universe of sequence homologs collected in (1).   3. Accurate sequence alignment Tools such as ClustalW (Chenna et al. 2003) are used to construct an accurate alignment of all the sequence homologs. The seed sequence is included in this alignment. This multiple sequence alignment establishes the equivalent positions of the PCS in each sequence homolog.   4. Function evaluation. The ligand binding properties of each of the aligned sequence homologs is determined by measuring their compliance with the PCS sequence filter. A “Hamming distance”, H, is assigned for each homolog, which specifies the degree of sequence identity of all the residues at the aligned PCS positions. A value of H=0 indicates that the identities of all the residues at the aligned PCS positions match the amino acid(s) allowed in the PCS search filter; H&gt;0, indicates that one or more aligned positions have disallowed residues. Sequences for which H=0 are predicted to encode binding proteins of the target ligand.   5. Selection of representative SAFE homologs. The sequence homologs are ordered by (a) identity with the seed PCS, as measured by the Hamming distance, (b) fractional overall sequence identity with the seed sequence. A subset for sequences with H=0, sampling the fractional overall sequence identity is selected for experimental verification.
 
These steps are encoded in the ProteinHunter software tool, which encodes the flow of execution, applies the PCS search filter, and visualizes the results, and handles organism annotations such as thermophilicity, and Gram stain status.
       

     The ProteinHunter package always executes BLAST searches, with the following command 
     “blastall -p blastp -m 8 -b 50000 -d % s -i&lt;INPUT FILE&gt; -o&lt;OUTPUT FILE&gt;” 
     where &lt;INPUT FILE&gt; and &lt;OUTPUT FILE&gt; specify the input and output files, respectively for a given calculation. This command executes the BLAST alignment program for protein sequences with default parameters, intrinsically set by the program. The BLAST program version is 2.2.24. 
     The ProteinHunter package always executes multiple sequence alignments with the following command 
     “clustalw -infile=&lt;INPUT FILE&gt; -outfile=&lt;OUTPUTFILE&gt; -align -quiet” 
     This command executes the CLUSTALW multi-sequence alignment program for protein sequences. There are no user-specified parameter settings that alter the alignment behavior of the program. The CLUSTALW program version is 2.1. 
     Annotated genomic and plasmid sequences of 5062 prokaryotes were obtained from the National Center of Biotechnology Information (ftp://ftp.ncbi.nih.gov/genomes/Bacteriaall.gbk.tar.gz). 
     Identification of Ca II —HCO 3  Binding Proteins. 
     The cyanobacterium  Synechocystis  PCC 6803 has a bicarbonate PBP (synBicarbBP1) (Omata 1999) which is a component of an uptake system that builds up high cytoplasmic CO 2  concentrations for photosynthetic carbon fixation (Price 2008). The X-ray structure of this protein revealed that bicarbonate binds as a Ca 2+  complex (Koropatkin 2007) ( FIGS. 3A  and B). This structure was used to define a PCS encoding recognition of the Ca II —HCO 3  complex ( FIGS. 3B-C ). Two of the bicarbonate oxygens participate in Ca 2+  coordination; the other oxygen forms hydrogen bonds with T192 and W99 ( FIG. 3B ). The Ca 2+  ion coordination sphere is a septa-coordinate pentagonal bipyramid. A PCS filter specifying the amino acid identity at each of these seven positions was used to predict Ca II —HCO 3  binding proteins. The synBicarbBP1 sequence was used as a seed to identify 742 sequence homologs in 484 replicons, representing 289 different species. Of these 38 were predicted to be Ca II —HCO 3  binding proteins based on their PCS (Table 1). The overall sequence identities of these homologs relative to the seed varied from 100% to 61%. 
     
       
         
           
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 synBicarbBP1 homologs. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 # 
                 Name 
                 70 
                 99 
                 121 
                 152 
                 192 
                 198 
               
               
                   
               
               
                 1 
                 2I4C| 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 2 
                 NC_017052|YP_005410477.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 3 
                 NC_017038|YP_005384601.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 4 
                 NC_017039|YP_005387770.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 5 
                 NC_000911|NP_442732.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 6 
                 NC_010546|YP_001801722.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 7 
                 NC_011729|YP_002379906.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 8 
                 NC_019703|YP_007108955.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 9 
                 NC_014501|YP_003887015.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 10 
                 NC_019748|YP_007133714.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 11 
                 NC_004113|NP_682790.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 12 
                 NC_007776|YP_476857.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 13 
                 NC_007775|YP_474359.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 14 
                 NC_023033|NK55_06690 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 15 
                 NC_010296|YP_001657013.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 16 
                 NC_019745|YP_007129075.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 17 
                 NC_019689|YP_007080477.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 18 
                 NC_019680|YP_007062720.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 19 
                 NC_019729|YP_007115550.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 20 
                 NC_019676|YP_007049841.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 21 
                 NC_013161|YP_003138293.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 22 
                 NC_019695|YP_007090308.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 23 
                 NC_010628|YP_001865867.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 24 
                 NC_011726|YP_002373642.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 25 
                 NC_007413|YP_321546.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 26 
                 NC_019738|YP_007123904.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 27 
                 NC_019684|YP_007076410.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 28 
                 NC_003272|NP_486917.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 29 
                 NC_019682|YP_007066695.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 30 
                 NC_019757|YP_007149519.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 31 
                 NC_019702|YP_007104545.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 32 
                 NC_007604|YP_400505.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 33 
                 NC_006576|YP_173184.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 34 
                 NC_011884|YP_002485585.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 35 
                 NC_019771|YP_007158727.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 36 
                 NC_019751|YP_007137061.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 37 
                 NC_019675|YP_007045476.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 38 
                 NC_019697|YP_007099445.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                 39 
                 NC_019439|YP_007001023.1 
                 E 
                 W 
                 Q 
                 N 
                 T 
                 Q 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 # 
                 270 
                 271 
                 Identity 
                 Gram 
                 Temperature 
                 Organism 
               
               
                   
               
               
                 1 
                 E 
                 E 
               
               
                 2 
                 E 
                 E 
                 1 
                 − 
                 Mesophilic 
                   Synechocystis  sp. 
               
               
                 3 
                 E 
                 E 
                 1 
                 − 
                 Mesophilic 
                   Synechocystis  sp. 
               
               
                 4 
                 E 
                 E 
                 1 
                 − 
                 Mesophilic 
                   Synechocystis  sp. 
               
               
                 5 
                 E 
                 E 
                 1 
                 − 
                 Mesophilic 
                   Synechocystis  sp. 
               
               
                 6 
                 E 
                 E 
                 0.84 
                 − 
                 Mesophilic 
                   Cyanothece  sp. 
               
               
                 7 
                 E 
                 E 
                 0.83 
                 − 
                 Mesophilic 
                   Cyanothece  sp. 
               
               
                 8 
                 E 
                 E 
                 0.8 
                 + 
                 Mesophilic 
                   Geitlerinema  sp. 
               
               
                 9 
                 E 
                 E 
                 0.8 
                 − 
                 Mesophilic 
                   Cyanothece  sp. 
               
               
                 10 
                 E 
                 E 
                 0.73 
                 + 
                 Mesophilic 
                 
                   Stanieria cyanosphaera 
                 
               
               
                 11 
                 E 
                 E 
                 0.72 
                 − 
                 Thermophilic 
                 
                   Thermosynechococcus elongatus 
                 
               
               
                 12 
                 E 
                 E 
                 0.72 
                 − 
                 Thermophilic 
                   Synechococcus  sp. 
               
               
                 13 
                 E 
                 E 
                 0.71 
                 − 
                 Thermophilic 
                   Synechococcus  sp. 
               
               
                 14 
                 E 
                 E 
                 0.71 
                 − 
                 Mesophilic 
                   Thermosynechococcus  sp. 
               
               
                 15 
                 E 
                 E 
                 0.7 
                 − 
                 Mesophilic 
                 
                   Microcystis aeruginosa 
                 
               
               
                 16 
                 E 
                 E 
                 0.69 
                 + 
                 Mesophilic 
                   Gloeocapsa  sp. 
               
               
                 17 
                 E 
                 E 
                 0.69 
                 + 
                 Mesophilic 
                   Pleurocapsa  sp. 
               
               
                 18 
                 E 
                 E 
                 0.69 
                 − 
                 Thermophilic 
                   Synechococcus  sp. 
               
               
                 19 
                 E 
                 E 
                 0.68 
                 + 
                 Mesophilic 
                   Oscillatoria nigro - viridis   
               
               
                 20 
                 E 
                 E 
                 0.68 
                 − 
                 Mesophilic 
                   Nostoc  sp. 
               
               
                 21 
                 E 
                 E 
                 0.68 
                 − 
                 Mesophilic 
                   Cyanothece  sp. 
               
               
                 22 
                 E 
                 E 
                 0.67 
                 + 
                 Mesophilic 
                 
                   Chroococcidiopsis thermalis 
                 
               
               
                 23 
                 E 
                 E 
                 0.67 
                 − 
                 Mesophilic 
                 
                   Nostoc punctiforme 
                 
               
               
                 24 
                 E 
                 E 
                 0.66 
                 − 
                 Mesophilic 
                   Cyanothece  sp. 
               
               
                 25 
                 E 
                 E 
                 0.66 
                 + 
                 Mesophilic 
                 
                   Anabaena variabilis 
                 
               
               
                 26 
                 E 
                 E 
                 0.66 
                 + 
                 Mesophilic 
                   Microcoleus  sp. 
               
               
                 27 
                 E 
                 E 
                 0.66 
                 − 
                 Mesophilic 
                   Nostoc  sp. 
               
               
                 28 
                 E 
                 E 
                 0.66 
                 − 
                 Mesophilic 
                   Nostoc  sp. 
               
               
                 29 
                 E 
                 E 
                 0.65 
                 + 
                 Mesophilic 
                   Calothrix  sp. 
               
               
                 30 
                 E 
                 E 
                 0.64 
                 + 
                 Mesophilic 
                 
                   Cylindrospermum stagnale 
                 
               
               
                 31 
                 E 
                 E 
                 0.64 
                 − 
                 Thermophilic 
                   Synechococcus  sp. 
               
               
                 32 
                 E 
                 E 
                 0.64 
                 − 
                 Mesophilic 
                 
                   Synechococcus elongatus 
                 
               
               
                 33 
                 E 
                 E 
                 0.64 
                 − 
                 Mesophilic 
                 
                   Synechococcus elongatus 
                 
               
               
                 34 
                 E 
                 E 
                 0.63 
                 − 
                 Mesophilic 
                   Cyanothece  sp. 
               
               
                 35 
                 E 
                 E 
                 0.63 
                 + 
                 Mesophilic 
                 
                   Anabaena cylindrica 
                 
               
               
                 36 
                 E 
                 E 
                 0.63 
                 + 
                 Mesophilic 
                   Calothrix  sp. 
               
               
                 37 
                 E 
                 E 
                 0.62 
                 + 
                 Mesophilic 
                 
                   Cyanobium gracile 
                 
               
               
                 38 
                 E 
                 E 
                 0.61 
                 + 
                 Mesophilic 
                 
                   Chamaesiphon minutus 
                 
               
               
                 39 
                 E 
                 E 
                 0.61 
                 + 
                 Mesophilic 
                   Anabaena  sp. 
               
               
                   
               
            
           
         
       
     
     Identification of Fe II —HCO 3  Binding Proteins. 
     Structures have been determined for Fe III —HCO 3  binding proteins from  Mannheimia haemolytica  (mhFeBP1) and  Thermus thermophilus  (ttFeBP5) ( FIG. 4 ). These revealed that two of the bicarbonate oxygens form a bidentate interaction with the bound iron. The other oxygen forms hydrogen bonds with an arginine. A second arginine also interacts with the bound bicarbonate. The Fe III  coordination sphere is complete by the phenolic hydroxyls of three tyrosines. In both proteins two of the three tyrosines are located adjacent to each on a helix (helix A), and the third tyrosine is contributed by a second helix (helix B). The location of this tyrosine within the B helix differs by a ½-helical turn in the two proteins. Accordingly, the positional information is slightly different for the PCS filters of these proteins, even though the compositional information is identical ( FIG. 4C ). 
     The mhFeBP1 sequence was used as a seed to identify 1290 sequence homologs in 958 replicons, representing 601 different species. Of these 522 were predicted to be Fe III —HCO 3  binding proteins based on their PCS (Table 2). Using the ttFeBP5 seed sequences, 1512 homologs (1172 replicons, 626 species) were identified, of which 70 satisfied the PCS filter (Table 3). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 mhFeBP1 homologs. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 # 
                 Name 
                 10 
                 101 
                 142 
                 198 
                 199 
                 Identity 
                 Gram 
                 Temperature 
                 Organism 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 1SI0| 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                   
                   
                   
                   
               
               
                 2 
                 NC_020834|YP_007669069.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 1 
                 + 
                 Mesophilic 
                 
                   Mannheimia haemolytica 
                 
               
               
                 3 
                 NC_020833|YP_007666187.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 1 
                 + 
                 Mesophilic 
                 
                   Mannheimia haemolytica 
                 
               
               
                 4 
                 NC_021082|YP_007884192.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 1 
                 + 
                 Mesophilic 
                 
                   Mannheimia haemolytica 
                 
               
               
                 5 
                 NC_021743|F382_02715 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 1 
                 + 
                 Mesophilic 
                 
                   Mannheimia haemolytica 
                 
               
               
                 6 
                 NC_021883|N220_08810 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 1 
                 + 
                 Mesophilic 
                 
                   Mannheimia haemolytica 
                 
               
               
                 7 
                 NC_021739|J451_03020 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 1 
                 + 
                 Mesophilic 
                 
                   Mannheimia haemolytica 
                 
               
               
                 8 
                 NC_021738|J450_02175 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.98 
                 + 
                 Mesophilic 
                 
                   Mannheimia haemolytica 
                 
               
               
                 9 
                 NC_011852|YP_002475690.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.86 
                 − 
                 Mesophilic 
                 
                   Haemophilus parasuis 
                 
               
               
                 10 
                 NC_020515|YP_007547693.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.84 
                 + 
                 Mesophilic 
                 
                   Bibersteinia trehalosi 
                 
               
               
                 11 
                 NC_017027|YP_005363665.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.8 
                 − 
                 Mesophilic 
                 
                   Pasteurella multocida 
                 
               
               
                 12 
                 NC_002663|NP_244988.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.8 
                 − 
                 Mesophilic 
                 
                   Pasteurella multocida 
                 
               
               
                 13 
                 NC_017764|YP_006240219.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.8 
                 − 
                 Mesophilic 
                 
                   Pasteurella multocida 
                 
               
               
                 14 
                 NC_016808|YP_005177128.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.8 
                 − 
                 Mesophilic 
                 
                   Pasteurella multocida 
                 
               
               
                 15 
                 NC_010519|YP_001784574.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.78 
                 − 
                 Mesophilic 
                 
                   Haemophilus somnus 
                 
               
               
                 16 
                 NC_015460|YP_004419395.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.78 
                 − 
                 Mesophilic 
                 
                   Gallibacterium anatis 
                 
               
               
                 17 
                 NC_008309|YP_718993.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.78 
                 − 
                 Mesophilic 
                 
                   Haemophilus somnus 
                 
               
               
                 18 
                 NC_022528|YP_008621989.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.64 
                 − 
                 Mesophilic 
                 
                   Vibrio nigripulchritudo 
                 
               
               
                 19 
                 NC_022349|YP_008535958.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.64 
                 − 
                 Mesophilic 
                 
                   Vibrio alginolyticus 
                 
               
               
                 20 
                 NC_013456|YP_003285201.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.64 
                 − 
                 Mesophilic 
                   Vibrio  sp. 
               
               
                 21 
                 NC_006370|YP_131292.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.64 
                 − 
                 Psychrophilic 
                 
                   Photobacterium profundum 
                 
               
               
                 22 
                 NC_011312|YP_002263934.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.63 
                 − 
                 Psychrophilic 
                 
                   Aliivibrio salmonicida 
                 
               
               
                 23 
                 NC_009783|YP_001446607.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.63 
                 − 
                 Mesophilic 
                 
                   Vibrio campbellii 
                 
               
               
                 24 
                 NC_022223|N175_12595 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.63 
                 + 
                 Mesophilic 
                 
                   Listonella anguillarum 
                 
               
               
                 25 
                 NC_019955|YP_007275951.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.63 
                 − 
                 Mesophilic 
                 
                   Vibrio parahaemolyticus 
                 
               
               
                 26 
                 NC_020802|YP_007640830.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.63 
                 + 
                 Mesophilic 
                   Psychromonas  sp. 
               
               
                 27 
                 NC_015633|YP_004567100.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.63 
                 − 
                 Mesophilic 
                 
                   Vibrio anguillarum 
                 
               
               
                 28 
                 NC_021848|M636_09480 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.63 
                 − 
                 Mesophilic 
                 
                   Vibrio parahaemolyticus 
                 
               
               
                 29 
                 NC_021847|M634_4850 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.63 
                 − 
                 Mesophilic 
                 
                   Vibrio parahaemolyticus 
                 
               
               
                 30 
                 NC_004603|NP_798870.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.63 
                 − 
                 Mesophilic 
                 
                   Vibrio parahaemolyticus 
                 
               
               
                 31 
                 NC_011753|YP_002418100.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.63 
                 − 
                 Mesophilic 
                 
                   Vibrio splendidus 
                 
               
               
                 32 
                 NC_011184|YP_002156950.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.62 
                 − 
                 Mesophilic 
                 
                   Vibrio fischeri 
                 
               
               
                 33 
                 NC_006840|VF2151 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.62 
                 − 
                 Mesophilic 
                 
                   Vibrio fischeri 
                 
               
               
                 34 
                 NC_016613|YP_005024030.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.62 
                 − 
                 Mesophilic 
                   Vibrio  sp. 
               
               
                 35 
                 NC_017270|YP_005633217.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.62 
                 − 
                 Mesophilic 
                 
                   Vibrio cholerae 
                 
               
               
                 36 
                 NC_012578|YP_002809340.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.62 
                 − 
                 Mesophilic 
                 
                   Vibrio cholerae 
                 
               
               
                 37 
                 NC_012668|YP_002879527.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.62 
                 − 
                 Mesophilic 
                 
                   Vibrio cholerae 
                 
               
               
                 38 
                 NC_016445|YP_004936187.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.62 
                 − 
                 Mesophilic 
                 
                   Vibrio cholerae 
                 
               
               
                 39 
                 NC_016944|YP_005332437.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.62 
                 − 
                 Mesophilic 
                 
                   Vibrio cholerae 
                 
               
               
                 40 
                 NC_002505|NP_230257.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.62 
                 − 
                 Mesophilic 
                 
                   Vibrio cholerae 
                 
               
               
                 41 
                 NC_009457|YP_001216102.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.62 
                 − 
                 Mesophilic 
                 
                   Vibrio cholerae 
                 
               
               
                 42 
                 NC_014965|YP_004187783.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.61 
                 − 
                 Mesophilic 
                 
                   Vibrio vulnificus 
                 
               
               
                 43 
                 NC_005139|NP_935539.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.61 
                 − 
                 Mesophilic 
                 
                   Vibrio vulnificus 
                 
               
               
                 44 
                 NC_004459|NP_760550.2 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.61 
                 − 
                 Mesophilic 
                 
                   Vibrio vulnificus 
                 
               
               
                 45 
                 NC_016602|YP_004992111.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.6 
                 − 
                 Mesophilic 
                 
                   Vibrio furnissii 
                 
               
               
                 46 
                 NC_008709|YP_944308.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.6 
                 + 
                 Psychrophilic 
                 
                   Psychromonas ingrahamii 
                 
               
               
                 47 
                 NC_015424|YP_004394158.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.6 
                 − 
                 Mesophilic 
                 
                   Aeromonas veronii 
                 
               
               
                 48 
                 NC_012691|YP_002893739.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.59 
                 − 
                 Mesophilic 
                 
                   Tolumonas auensis 
                 
               
               
                 49 
                 NC_021290|YP_008041799.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.59 
                 − 
                 Mesophilic 
                 
                   Aeromonas hydrophila 
                 
               
               
                 50 
                 NC_008570|YP_855192.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.59 
                 − 
                 Mesophilic 
                 
                   Aeromonas hydrophila 
                 
               
               
                 51 
                 NC_016745|YP_005093558.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.59 
                 + 
                 Mesophilic 
                   Oceanimonas  sp. 
               
               
                 52 
                 NC_009348|YP_001140561.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.59 
                 − 
                 Mesophilic. 
                 
                   Aeromonas salmonicida 
                 
               
               
                 53 
                 NC_014541|YP_003914651.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.57 
                 − 
                 Mesophilic 
                 
                   Ferrimonas balearica 
                 
               
               
                 54 
                 NC_010334|YP_001673131.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.57 
                 − 
                 Psychrophilic 
                 
                   Shewanella halifaxensis 
                 
               
               
                 55 
                 NC_008345|YP_749334.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.57 
                 − 
                 Mesophilic 
                 
                   Shewanella frigidimarina 
                 
               
               
                 56 
                 NC_010506|YP_001759384.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.56 
                 − 
                 Mesophilic 
                 
                   Shewanelia woodyi 
                 
               
               
                 57 
                 NC_009901|YP_001500706.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.56 
                 − 
                 Mesophilic 
                 
                   Shewanella pealeana 
                 
               
               
                 58 
                 NC_008700|YP_926544.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.56 
                 − 
                 Mesophilic 
                 
                   Shewaneiia amazonensis 
                 
               
               
                 59 
                 NC_014012|YP_003555527.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.55 
                 − 
                 Psychrophilic 
                 
                   Shewanella violacea 
                 
               
               
                 60 
                 NC_009092|YP_001092992.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.55 
                 − 
                 Mesophilic 
                 
                   Shewaneiia loihica 
                 
               
               
                 61 
                 NC_009831|YP_001472684.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.55 
                 − 
                 Psychrophilic 
                 
                   Shewanella sediminis 
                 
               
               
                 62 
                 NC_007645|YP_435624.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.55 
                 − 
                 Mesophilic 
                 
                   Hahella chejuensis 
                 
               
               
                 63 
                 NC_007481|YP_341153.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.55 
                 − 
                 Psychrophilic 
                 
                   Pseudoalteromonas haloplanktis 
                 
               
               
                 64 
                 NC_008321|YP_735470.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.55 
                 − 
                 Mesophilic 
                   Shewanella  sp. 
               
               
                 65 
                 NC_007643|Rru_A0769 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.55 
                 − 
                 Mesophilic 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 66 
                 NC_008322|YP_736666.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.55 
                 − 
                 Mesophilic 
                   Shewanella  sp. 
               
               
                 67 
                 NC_017584|YP_006047056.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.55 
                 − 
                 Mesophilic 
                 
                   Rhodospirillum rubrum 
                 
               
               
                 68 
                 NC_004347|SO_0744 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.54 
                 − 
                 Mesophilic 
                 
                   Shewanella oneidensis 
                 
               
               
                 69 
                 NC_008577|YP_871146.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.54 
                 − 
                 Mesophilic 
                   Shewanella  sp. 
               
               
                 70 
                 NC_014803|YP_004069765.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.54 
                 − 
                 Psychrophilic 
                   Pseudoalteromonas  sp. 
               
               
                 71 
                 NC_009997|YP_001556237.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.54 
                 − 
                 Mesophilic 
                 
                   Shewanella baltica 
                 
               
               
                 72 
                 NC_016901|YP_005275030.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.54 
                 − 
                 Mesophilic 
                 
                   Shewanella baltica 
                 
               
               
                 73 
                 NC_003910|YP_267761.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.53 
                 − 
                 Psychrophilic 
                 
                   Colwellia psychrerythraea 
                 
               
               
                 74 
                 NC_017571|YP_006022146.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.53 
                 − 
                 Mesophilic 
                 
                   Shewanella baltica 
                 
               
               
                 75 
                 NC_011663|YP_002359536.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.53 
                 − 
                 Mesophilic 
                 
                   Shewanella baltica 
                 
               
               
                 76 
                 NC_012587|YP_002824894.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.53 
                 − 
                 Mesophilic 
                 
                   Sinorhizobium fredii 
                 
               
               
                 77 
                 NC_016812|YP_005187712.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.53 
                 − 
                 Mesophilic 
                 
                   Sinorhizobium fredii 
                 
               
               
                 78 
                 NC_009665|YP_001367881.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.53 
                 − 
                 Mesophilic 
                 
                   Shewanella baltica 
                 
               
               
                 79 
                 NC_009636|YP_001326020.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.53 
                 − 
                 Mesophilic 
                 
                   Sinorhizobium medicae 
                 
               
               
                 80 
                 NC_017506|YP_005885148.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.53 
                 − 
                 ? 
                 
                   Marinobacter adhaerens 
                 
               
               
                 81 
                 NC_017322|YP_005712524.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.53 
                 − 
                 Mesophilic 
                 
                   Sinorhizobium meliloti 
                 
               
               
                 82 
                 NC_020528|SM2011_c00784 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.53 
                 − 
                 Mesophilic 
                 
                   Sinorhizobium meliloti 
                 
               
               
                 83 
                 NC_018000|YP_006395735.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.53 
                 − 
                 Mesophilic 
                 
                   Sinorhizobium fredii 
                 
               
               
                 84 
                 NC_018700|YP_006839225.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.53 
                 − 
                 Mesophilic 
                 
                   Sinorhizobium meliloti 
                 
               
               
                 85 
                 NC_018268|YP_006557993.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.53 
                 − 
                 Mesophilic 
                   Marinobacter  sp. 
               
               
                 86 
                 NC_011566|VP_002313357.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.52 
                 − 
                 Mesophilic 
                 
                   Shewanella piezotolerans 
                 
               
               
                 87 
                 NC_017325|YP_005718919.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.52 
                 − 
                 Mesophilic 
                 
                   Sinorhizobium meliloti 
                 
               
               
                 88 
                 NC_015590|YP_004547809.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.52 
                 − 
                 Mesophilic 
                 
                   Sinorhizobium meliloti 
                 
               
               
                 89 
                 NC_019845|C770_GR4Chr0719 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.52 
                 − 
                 Mesophilic 
                 
                   Sinorhizobium meliloti 
                 
               
               
                 90 
                 NC_017964|YP_006379465.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.52 
                 + 
                 Mesophilic 
                 
                   Advenella kashmirensis 
                 
               
               
                 91 
                 NC_003047|NP_384835.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.52 
                 − 
                 Mesophilic 
                 
                   Sinorhizobium meliloti 
                 
               
               
                 92 
                 NC_008750|YP_962119.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.52 
                 − 
                 Mesophilic 
                   Shewanella  sp. 
               
               
                 93 
                 NC_017566|YP_006011216.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.52 
                 − 
                 Mesophilic 
                 
                   Shewanella putrefaciens 
                 
               
               
                 94 
                 NC_009438|YP_001184738.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.52 
                 − 
                 Mesophilic 
                 
                   Shewanella putrefaciens 
                 
               
               
                 95 
                 NC_015259|YP_004305240.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.52 
                 − 
                 Mesophilic 
                 
                   Polymorphum gilvum 
                 
               
               
                 96 
                 NC_008740|YP_958371.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.52 
                 − 
                 Mesophilic 
                 
                   Marinobacter aquaeolei 
                 
               
               
                 97 
                 NC_016642|YP_005079787.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.52 
                 + 
                 Mesophilic 
                   Pseudovibrio  sp. 
               
               
                 98 
                 NC_017067|YP_005430089.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.52 
                 − 
                 Mesophilic 
                 
                   Marinobacter hydrocarbonoclast 
                 
               
               
                 99 
                 NC_008254|YP_672740.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.52 
                 − 
                 Mesophilic 
                   Chelativorans  sp. 
               
               
                 100 
                 NC_005296|NP_949488.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.52 
                 − 
                 Mesophilic 
                 
                   Rhodopseudomonas palustris 
                 
               
               
                 101 
                 NC_022535|YP_008631147.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.51 
                 + 
                 Mesophilic 
                   Rhizobium  sp. 
               
               
                 102 
                 NC_011004|YP_001993597.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.51 
                 − 
                 Mesophilic 
                 
                   Rhodopseudomonas palustris 
                 
               
               
                 103 
                 NC_015183|YP_004277506.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.51 
                 + 
                 Mesophilic 
                   Agrobacterium  sp. 
               
               
                 104 
                 NC_007493|RSP_2913 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.51 
                 − 
                 Mesophilic 
                 
                   Rhodobacter sphaeroides 
                 
               
               
                 105 
                 NC_003062|NP_353236.2 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.51 
                 + 
                 Mesophilic 
                 
                   Agrobacterium fabrum 
                 
               
               
                 106 
                 NC_009049|YP_001043439.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.51 
                 − 
                 Mesophilic 
                 
                   Rhodobacter sphaeroides 
                 
               
               
                 107 
                 NC_011963|YP_002525591.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.51 
                 − 
                 Mesophilic 
                 
                   Rhodobacter sphaeroides 
                 
               
               
                 108 
                 NC_021033|YP_007821259.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.51 
                 − 
                 Mesophilic 
                 
                   Taylorella asinigenitalis 
                 
               
               
                 109 
                 NC_021036|YP_007825629.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.51 
                 − 
                 Mesophilic 
                 
                   Taylorella equigenitalis 
                 
               
               
                 110 
                 NC_014914|YP_004129165.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.51 
                 − 
                 Mesophilic 
                 
                   Taylorella equigenitalis 
                 
               
               
                 111 
                 NC_018108|YP_006502736.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.51 
                 − 
                 Mesophilic 
                 
                   Taylorella equigenitalis 
                 
               
               
                 112 
                 NC_009428|YP_001167314.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.51 
                 − 
                 Mesophilic 
                 
                   Rhodobacter sphaeroides 
                 
               
               
                 113 
                 NC_011386|YP_002288119.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.5 
                 − 
                 Mesophilic 
                 
                   Oligotropha carboxidovorans 
                 
               
               
                 114 
                 NC_017538|YP_005951842.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.5 
                 − 
                 Mesophilic 
                 
                   Oligotropha carboxidovorans 
                 
               
               
                 115 
                 NC_016043|YP_004874880.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.5 
                 − 
                 Mesophilic 
                 
                   Taylorella asinigenitalis 
                 
               
               
                 116 
                 NC_010511|YP_001772582.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.49 
                 − 
                 Mesophilic 
                   Methylobacterium  sp. 
               
               
                 117 
                 NC_007778|YP_485084.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.49 
                 − 
                 Mesophilic 
                 
                   Rhodopseudomonas palustris 
                 
               
               
                 118 
                 NC_014834|YP_004110640.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.49 
                 − 
                 Mesophilic 
                 
                   Rhodopseudomonas palustris 
                 
               
               
                 119 
                 NC_002927|NP_889482.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.49 
                 − 
                 Mesophilic 
                 
                   Bordetella bronchiseptica 
                 
               
               
                 120 
                 NC_018828|YP_006896532.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.49 
                 − 
                 Mesophilic 
                 
                   Bordetella parapertussis 
                 
               
               
                 121 
                 NC_017223|YP_005589733.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.48 
                 − 
                 Mesophilic 
                 
                   Bordetella pertussis 
                 
               
               
                 122 
                 NC_007958|YP_568577.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.48 
                 − 
                 Mesophilic 
                 
                   Rhodopseudomonas palustris 
                 
               
               
                 123 
                 NC_018518|YP_006626426.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.48 
                 − 
                 Mesophilic 
                 
                   Bordetella pertussis 
                 
               
               
                 124 
                 NC_002929|NP_880337.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.48 
                 − 
                 Mesophilic 
                 
                   Bordetella pertussis 
                 
               
               
                 125 
                 NC_002928|NP_885167.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.48 
                 − 
                 Mesophilic 
                 
                   Bordetella parapertussis 
                 
               
               
                 126 
                 NC_019382|YP_006966829.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.48 
                 − 
                 Mesophilic 
                 
                   Bordetella bronchiseptica 
                 
               
               
                 127 
                 NC_018829|YP_006900438.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.48 
                 − 
                 Mesophilic 
                 
                   Bordetella bronchiseptica 
                 
               
               
                 128 
                 NC_007925|YP_533792.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.48 
                 − 
                 Mesophilic 
                 
                   Rhodopseudomonas palustris 
                 
               
               
                 129 
                 NC_008435|YP_782979.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.48 
                 − 
                 Mesophilic 
                 
                   Rhodopseudomonas palustris 
                 
               
               
                 130 
                 NC_011894|YP_002501458.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.48 
                 − 
                 Mesophilic 
                 
                   Methylobacterium nodulans 
                 
               
               
                 131 
                 NC_017283|YP_005661039.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.48 
                 − 
                 Mesophilic 
                 
                   Brucella melitensis 
                 
               
               
                 132 
                 NC_010551|YP_001807800.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.48 
                 − 
                 Mesophilic 
                 
                   Burkholderia ambifaria 
                 
               
               
                 133 
                 NC_015858|YP_004757945.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.48 
                 − 
                 Mesophilic 
                 
                   Brucella pinnipedialis 
                 
               
               
                 134 
                 NC_014034|YP_003578715.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.48 
                 − 
                 Mesophilic 
                 
                   Rhodobacter capsulatus 
                 
               
               
                 135 
                 NC_018513|YP_006616387.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.48 
                 − 
                 Mesophilic 
                 
                   Burkholderia cepacia 
                 
               
               
                 136 
                 NC_009504|YP_001257663.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.48 
                 − 
                 Mesophilic 
                 
                   Brucella ovis 
                 
               
               
                 137 
                 NC_008390|YP_772986.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.48 
                 − 
                 Mesophilic 
                 
                   Burkholderia ambifaria 
                 
               
               
                 138 
                 NC_009668|YP_001372359.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.48 
                 − 
                 Mesophilic 
                 
                   Ochrobactrum anthropi 
                 
               
               
                 139 
                 NC_012442|YP_002734445.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Brucella melitensis 
                 
               
               
                 140 
                 NC_022535|YP_008631357.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 + 
                 Mesophilic 
                   Rhizobium  sp. 
               
               
                 141 
                 NC_017247|YP_005602286.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Brucella melitensis 
                 
               
               
                 142 
                 NC_013118|YP_003105477.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Brucella microti 
                 
               
               
                 143 
                 NC_017245|YP_005598924.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Brucella melitensis 
                 
               
               
                 144 
                 NC_010104|YP_001594643.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Brucella canis 
                 
               
               
                 145 
                 NC_010167|YP_001622484.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Brucella suis 
                 
               
               
                 146 
                 NC_010740|YP_001932452.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Brucella abortus 
                 
               
               
                 147 
                 NC_022906|V910_200569 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Brucella ceti 
                 
               
               
                 148 
                 NC_016777|YP_005114137.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Brucella abortus 
                 
               
               
                 149 
                 NC_016796|YP_005152703.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Brucella canis 
                 
               
               
                 150 
                 NC_007624|YP_418727.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Brucella abortus 
                 
               
               
                 151 
                 NC_006933|YP_223309.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Brucella abortus 
                 
               
               
                 152 
                 NC_003318|NP_541562.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Brucella melitensis 
                 
               
               
                 153 
                 NC_008687|YP_918207.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 + 
                 Mesophilic 
                 
                   Paracoccus denitrificans 
                 
               
               
                 154 
                 NC_010084|YP_001580275.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Burkholderia multivorans 
                 
               
               
                 155 
                 NC_009937|YP_001525161.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Azorhizobium caulinodans 
                 
               
               
                 156 
                 NC_010508|YP_001764480.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Burkholderia cenocepacia 
                 
               
               
                 157 
                 NC_015458|YP_004416353.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 ? 
                   Pusillimonas  sp. 
               
               
                 158 
                 NC_016775|YP_005108693.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Brucella suis 
                 
               
               
                 159 
                 NC_014217|YP_003692549.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Starkeya novella 
                 
               
               
                 160 
                 NC_007510|YP_368558.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Burkholderia lata 
                 
               
               
                 161 
                 NC_004311|NP_699880.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Brucella suis 
                 
               
               
                 162 
                 NC_011000|YP_002230239.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Burkholderia cenocepacia 
                 
               
               
                 163 
                 NC_022043|YP_008406497.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 + 
                 Mesophilic 
                 
                   Paracoccus aminophilus 
                 
               
               
                 164 
                 NC_008060|YP_620613.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Burkholderia cenocepacia 
                 
               
               
                 165 
                 NC_003062|NP_353437.2 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 + 
                 Mesophilic 
                 
                   Agrobacterium fabrum 
                 
               
               
                 166 
                 NC_008542|YP_834855.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Burkholderia cenocepacia 
                 
               
               
                 167 
                 NC_015183|YP_004277705.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 + 
                 Mesophilic 
                   Agrobacterium  sp. 
               
               
                 168 
                 NC_014640|YP_003979373.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.47 
                 − 
                 Mesophilic 
                 
                   Achromobacter xylosoxidans 
                 
               
               
                 169 
                 NC_023061|AX27061_2729 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Achromobacter xylosoxidans 
                 
               
               
                 170 
                 NC_021285|YP_008031617.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Achromobacter xylosoxidans 
                 
               
               
                 171 
                 NC_022659|BBK_250 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Burkholderia pseudomallei 
                 
               
               
                 172 
                 NC_014931|YP_004156555.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Variovorax paradoxus 
                 
               
               
                 173 
                 NC_014010|YP_003552419.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Candidatus Puniceispirillum 
                 
               
               
                 174 
                 NC_013446|CtCNB1_0418 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Comamonas testosteroni 
                 
               
               
                 175 
                 NC_021884|BDL_744 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Burkholderia pseudomallei 
                 
               
               
                 176 
                 NC_018527|YP_006652216.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Burkholderia pseudomallei 
                 
               
               
                 177 
                 NC_006348|YP_103391.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Burkholderia mallei 
                 
               
               
                 178 
                 NC_008785|YP_992517.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Burkholderia mallei 
                 
               
               
                 179 
                 NC_008836|YP_001026683.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Burkholderia mallei 
                 
               
               
                 180 
                 NC_009080|YP_001080034.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Burkholderia mallei 
                 
               
               
                 181 
                 NC_009076|YP_001065655.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Burkholderia pseudomallei 
                 
               
               
                 182 
                 NC_012695|YP_002896116.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Burkholderia pseudomallei 
                 
               
               
                 183 
                 NC_010170|YP_001630893.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Bordetella petrii 
                 
               
               
                 184 
                 NC_010645|YP_786440.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Bordetella avium 
                 
               
               
                 185 
                 NC_021173|YP_007919037.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Burkholderia thailandensis 
                 
               
               
                 186 
                 NC_016078|YP_004898234.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 + 
                 Mesophilic 
                 
                   Pelagibacterium halotolerans 
                 
               
               
                 187 
                 NC_007651|YP_443368.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Burkholderia thailandensis 
                 
               
               
                 188 
                 NC_007434|YP_332916.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Burkholderia pseudomallei 
                 
               
               
                 189 
                 NC_009074|YP_001058421.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Burkholderia pseudomallei 
                 
               
               
                 190 
                 NC_017831|YP_006275273.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.46 
                 − 
                 Mesophilic 
                 
                   Burkholderia pseudomallei 
                 
               
               
                 191 
                 NC_022247|VAPA_1c38240 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.45 
                 − 
                 Mesophilic 
                 
                   Variovorax paradoxus 
                 
               
               
                 192 
                 NC_012791|YP_002945581.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.45 
                 − 
                 Mesophilic 
                 
                   Variovorax paradoxus 
                 
               
               
                 193 
                 NC_010510|YP_001767013.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.45 
                 − 
                 Mesophilic 
                 
                   Methylobacterium radiotolerans 
                 
               
               
                 194 
                 NC_009720|YP_001418646.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.45 
                 − 
                 Mesophilic 
                 
                   Xanthobacter autotrophicus 
                 
               
               
                 195 
                 NC_017956|YP_006370956.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.45 
                 + 
                 Mesophilic 
                 
                   Tistrella mobilis 
                 
               
               
                 196 
                 NC_006350|YP_107899.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.45 
                 − 
                 Mesophilic 
                 
                   Burkholderia pseudomallei 
                 
               
               
                 197 
                 NC_010725|YP_001923256.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.45 
                 − 
                 Mesophilic 
                 
                   Methylobacterium populi 
                 
               
               
                 198 
                 NC_018525|YP_006646037.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.45 
                 − 
                 Mesophilic 
                 
                   Pectobacterium carotovorum 
                 
               
               
                 199 
                 NC_012917|YP_003016929.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.45 
                 − 
                 Mesophilic 
                 
                   Pectobacterium carotovorum 
                 
               
               
                 200 
                 NC_004547|YP_049576.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.45 
                 − 
                 Mesophilic 
                 
                   Pectobacterium atrosepticum 
                 
               
               
                 201 
                 NC_012988|YP_003066252.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.44 
                 − 
                 Mesophilic 
                 
                   Methylobacterium extorquens 
                 
               
               
                 202 
                 NC_012808|YP_002961615.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.44 
                 − 
                 Mesophilic 
                 
                   Methylobacterium extorquens 
                 
               
               
                 203 
                 NC_010172|YP_001638056.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.44 
                 − 
                 Mesophilic 
                 
                   Methylobacterium extorquens 
                 
               
               
                 204 
                 NC_010995|YP_001984073.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.44 
                 − 
                 Mesophilic 
                 
                   Cellvibrio japonicus 
                 
               
               
                 205 
                 NC_014166|YP_003656801.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.44 
                 − 
                 Mesophilic 
                 
                   Arcobacter nitrofigilis 
                 
               
               
                 206 
                 NC_011757|YP_002419429.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.44 
                 − 
                 Mesophilic 
                 
                   Methylobacterium extorquens 
                 
               
               
                 207 
                 NC_015061|YP_004212396.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.44 
                 + 
                 Mesophilic 
                   Rahnella  sp. 
               
               
                 208 
                 NC_017047|YP_005401406.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.44 
                 + 
                 Mesophilic 
                 
                   Rahnella aquatilis 
                 
               
               
                 209 
                 NC_017845|YP_006283928.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.44 
                 − 
                 Mesophilic 
                   Pectobacterium  sp. 
               
               
                 210 
                 NC_020418|YP_007504644.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.43 
                 + 
                 Mesophilic 
                 
                   Morganella morganii 
                 
               
               
                 211 
                 NC_022997|W911_04275 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.43 
                 + 
                 Mesophilic 
                 
                   Hyphomicrobium nitrativorans 
                 
               
               
                 212 
                 NC_017309|YP_005700102.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.43 
                 − 
                 Mesophilic 
                 
                   Edwardsiella tarda 
                 
               
               
                 213 
                 NC_020796|ETAC_3335 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.43 
                 − 
                 Mesophilic 
                 
                   Edwardsiella piscicida 
                 
               
               
                 214 
                 NC_013889|YP_003459550.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.43 
                 + 
                 Mesophilic 
                   Thioalkalivibrio  sp. 
               
               
                 215 
                 NC_013421|YP_003260342.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.43 
                 − 
                 Mesophilic 
                 
                   Pectobacterium wasabiae 
                 
               
               
                 216 
                 NC_018002|YP_006405228.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.43 
                 + 
                 Mesophilic 
                 
                   Sulfurospirillum barnesii 
                 
               
               
                 217 
                 NC_012779|YP_002934481.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.43 
                 − 
                 Mesophilic 
                 
                   Edwardsiella ictaluri 
                 
               
               
                 218 
                 NC_013508|YP_003296812.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.43 
                 − 
                 Mesophilic 
                 
                   Edwardsiella tarda 
                 
               
               
                 219 
                 NC_018644|YP_006757561.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.43 
                 + 
                 Mesophilic 
                 alpha proteobacterium 
               
               
                 220 
                 NC_017192|YP_005554689.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.42 
                 − 
                 Mesophilic 
                   Arcobacter  sp. 
               
               
                 221 
                 NC_003911|SPO3287 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.42 
                 − 
                 Mesophilic 
                 
                   Ruegeria pomeroyi 
                 
               
               
                 222 
                 NC_015726|YP_004684614.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.42 
                 − 
                 Mesophilic 
                 
                   Cupriavidus necator 
                 
               
               
                 223 
                 NC_008044|YP_612119.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.42 
                 − 
                 Mesophilic 
                   Ruegeria  sp. 
               
               
                 224 
                 NC_006513|YP_159824.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.42 
                 − 
                 Mesophilic 
                 
                   Aromatoleum aromaticum 
                 
               
               
                 225 
                 NC_022513|N234_0870 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.42 
                 − 
                 Mesophilic 
                 
                   Ralstonia pickettii 
                 
               
               
                 226 
                 NC_012673|YP_002886303.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.42 
                 + 
                 Thermophilic 
                   Exiguobacterium  sp. 
               
               
                 227 
                 NC_014759|YP_004053760.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.42 
                 − 
                 Mesophilic 
                 
                   Marivirga tractuosa 
                 
               
               
                 228 
                 NC_019902|YP_007215684.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.42 
                 + 
                 Mesophilic 
                 
                   Thioalkalivibrio nitratireduce 
                 
               
               
                 229 
                 NC_014216|YP_003691508.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.42 
                 − 
                 Mesophilic 
                 
                   Desulfurivibrio alkaliphilus 
                 
               
               
                 230 
                 NC_018018|YP_006433889.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.42 
                 + 
                 Mesophilic 
                 
                   Flexibacter litoralis 
                 
               
               
                 231 
                 NC_017668|YP_006182380.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.42 
                 + 
                 Mesophilic 
                 
                   Halobacillus halophilus 
                 
               
               
                 232 
                 NC_013854|YP_003448425.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 + 
                 Mesophilic 
                   Azospirillum  sp. 
               
               
                 233 
                 NC_007908|Rfer_2758 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 + 
                 Mesophilic 
                 
                   Albidiferax ferrireducens 
                 
               
               
                 234 
                 NC_006512|YP_155448.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 − 
                 Mesophilic 
                 
                   Idiomarina loihiensis 
                 
               
               
                 235 
                 NC_008781|YP_981845.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 − 
                 Mesophilic 
                 
                   Polaromonas naphthalenivorans 
                 
               
               
                 236 
                 NC_009253|YP_001112883.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 + 
                 Mesophilic 
                 
                   Desulfotomaculum reducens 
                 
               
               
                 237 
                 NC_016622|YP _005038613.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 + 
                 Mesophilic 
                 
                   Azospirillum lipoferum 
                 
               
               
                 238 
                 NC_021286|YP_008034563.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 − 
                 Mesophilic 
                 
                   Idiomarina loihiensis 
                 
               
               
                 239 
                 NC_018010|YP_006406798.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 + 
                 Mesophilic 
                 
                   Belliella baltica 
                 
               
               
                 240 
                 NC_008313|YP_725275.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 − 
                 Mesophilic 
                 
                   Ralstonia eutropha 
                 
               
               
                 241 
                 NC_008702|YP_933709.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 − 
                 Mesophilic 
                   Azoarcus  sp. 
               
               
                 242 
                 NC_019738|YP_007124478.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 + 
                 Mesophilic 
                   Microcoleus  sp. 
               
               
                 243 
                 NC_010528|YP_002004788.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 − 
                 Mesophilic 
                 
                   Cupriavidus taiwanensis 
                 
               
               
                 244 
                 NC_015276|YP_004311821.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 − 
                 Mesophilic 
                 
                   Marinomonas mediterranea 
                 
               
               
                 245 
                 NC_320908|YP_007698507.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 + 
                 Mesophilic 
                 
                   Octadecabacter arcticus 
                 
               
               
                 246 
                 NC_007953|YP_556393.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 − 
                 Mesophilic 
                 
                   Burkholderia xenovorans 
                 
               
               
                 247 
                 NC_023135|METH_00275 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 + 
                 Mesophilic 
                 
                   Leisingera methylohalidivorans 
                 
               
               
                 248 
                 NC_017187|YP_005539339.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 − 
                 Mesophilic 
                 
                   Arcobacter butzleri 
                 
               
               
                 249 
                 NC_009850|YP_001490892.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 − 
                 Mesophilic 
                 
                   Arcobacter butzleri 
                 
               
               
                 250 
                 NC_022759|CFT03427_0122 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 − 
                 Mesophilic 
                 
                   Campylobacter fetus 
                 
               
               
                 251 
                 NC_020514|YP_007544802.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 + 
                 Mesophilic 
                 
                   Glaciecola psychrophila 
                 
               
               
                 252 
                 NC_021878|YP_00833.1820.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 − 
                 Mesophilic 
                 
                   Arcobacter butzleri 
                 
               
               
                 253 
                 NC_014366|YP_003811659.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 + 
                 Mesophilic 
                 gamma proteobacterium 
               
               
                 254 
                 NC_007775|YP_475512.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 − 
                 Thermophilic 
                   Synechococcus  sp. 
               
               
                 255 
                 NC_007776|YP_478858.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 − 
                 Thermophilic 
                   Synechococcus  sp. 
               
               
                 256 
                 NC_018708|YP_006855088.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 − 
                 Mesophilic 
                   Acidovorax  sp. 
               
               
                 257 
                 NC_005295|YP_179998.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 − 
                 Mesophilic 
                 
                   Ehrlichia ruminantium 
                 
               
               
                 258 
                 NC_004842|YP_153489.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 − 
                 Mesophilic 
                 
                   Anaplasma marginale 
                 
               
               
                 259 
                 NC_008752|YP_971208.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 − 
                 Mesophilic 
                 
                   Acidovorax citrulli 
                 
               
               
                 260 
                 NC_023065|YP_008937446.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.41 
                 + 
                 Mesophilic 
                 
                   Magnetospirillum gryphiswalden 
                 
               
               
                 261 
                 NC_011992|YP_002553562.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Acidovorax ebreus 
                 
               
               
                 262 
                 NC_012026|YP_002563199.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Anaplasma marginale 
                 
               
               
                 263 
                 NC_022760|U370_00265 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Anaplasma marginale 
                 
               
               
                 264 
                 NC_015422|YP_004389134.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Alicycliphilus denitrificans 
                 
               
               
                 265 
                 NC_016041|YP_004871348.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 + 
                 Mesophilic 
                 
                   Glaciecola nitratireducens 
                 
               
               
                 266 
                 NC_014910|YP_004126179.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Alicycliphilus denitrificans 
                 
               
               
                 267 
                 NC_018012|YP_006413475.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 + 
                 Mesophilic 
                 
                   Thiocystis violascens 
                 
               
               
                 268 
                 NC_017856|YP_006293166.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 + 
                 Mesophilic 
                 
                   Methylophaga frappieri 
                 
               
               
                 269 
                 NC_007799|YP_507013.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Ehrlichia chaffeensis 
                 
               
               
                 270 
                 NC_002978|NP_966643.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Wolbachia endosymbiont 
                 
               
               
                 271 
                 NC_006831|YP_196049.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Ehrlichia ruminantium 
                 
               
               
                 272 
                 NC_008825|YP_001020568.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Methylibium petroleiphilum 
                 
               
               
                 273 
                 NC_008786|YP_997316.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Verminephrobacter eiseniae 
                 
               
               
                 274 
                 NC_008782|YP_985876.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                   Acidovorax  sp. 
               
               
                 275 
                 NC_009654|YP_001342590.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                   Martnomonas  sp. 
               
               
                 276 
                 NC_012039|Cla_1526 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Campylobacter lari 
                 
               
               
                 277 
                 NC_011901|YP_002512540.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 + 
                 Mesophilic 
                 
                   Thioalkalivibrio sulfidiphilus 
                 
               
               
                 278 
                 NC_010002|YP_001565757.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Delftia acidovorans 
                 
               
               
                 279 
                 NC_022784|U128_00245 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Anaplasma marginale 
                 
               
               
                 280 
                 NC_015138|YP_004234852.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Acidovorax avenae 
                 
               
               
                 281 
                 NC_015677|YP_004619583.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Ramlibacter tataouinensis 
                 
               
               
                 282 
                 NC_015563|YP_004487487.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                   Delftia  sp. 
               
               
                 283 
                 NC_016616|YP_005029105.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 + 
                 Mesophilic 
                 
                   Dechlorosoma suillim 
                 
               
               
                 284 
                 NC_017857|YP_006297460.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 + 
                 Mesophilic. 
                 
                   Methylophaga nitratireducentic 
                 
               
               
                 285 
                 NC_018721|YP_006868342.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 + 
                 Mesophilic 
                 
                   Psychroflexus torquis 
                 
               
               
                 286 
                 NC_023137|Gal_03312 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 + 
                 Mesophilic 
                 
                   Phaeobacter gallaeciensis 
                 
               
               
                 287 
                 NC_012416|YP_002727371.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                   Wolbachia  sp. 
               
               
                 288 
                 NC_019748|YP_007133065.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 + 
                 Mesophilic 
                 
                   Stanieria cyanosphaera 
                 
               
               
                 289 
                 NC_023035|L21SP2_0743 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Hyperthermophilic 
                   Spirochaeta  sp. 
               
               
                 290 
                 NC_020911|YP_007702830.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 + 
                 Mesophilic 
                 
                   Octadecabacter antarcticus 
                 
               
               
                 291 
                 NC_021089|YP_007889339.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Wolbachia endosymbiont 
                 
               
               
                 292 
                 NC_017039|YP_005385198.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                   Synechocystis  sp. 
               
               
                 293 
                 NC_017075|YP_005437539.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 + 
                 Mesophilic 
                 
                   Rubrivivax gelatinosus 
                 
               
               
                 294 
                 NC_014836|YP_004113366.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Desulfurispirillum indicum 
                 
               
               
                 295 
                 NC_014323|YP_003775309.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Herbaspirillum seropedicae 
                 
               
               
                 296 
                 NC_007354|YP_302777.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Ehrlichia canis 
                 
               
               
                 297 
                 NC_007204|YP_264829.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Psychrophilic 
                 
                   Psychrobacter arcticus 
                 
               
               
                 298 
                 NC_018286|YP_006564286.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 + 
                 Mesophilic 
                 
                   Phaeobacter gallaeciensis 
                 
               
               
                 299 
                 NC_018290|YP_006574608.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 + 
                 Mesophilic 
                 
                   Phaeobacter inhibens 
                 
               
               
                 300 
                 NC_000911|NP_440162.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                   Synechocystis  sp. 
               
               
                 301 
                 NC_009138|YP_001099462.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.4 
                 − 
                 Mesophilic 
                 
                   Herminiimonas arsenicoxydans 
                 
               
               
                 302 
                 NC_012491|YP_002770441.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 + 
                 Mesophilic 
                 
                   Brevibacillus brevis 
                 
               
               
                 303 
                 NC_010981|YP_001975438.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Mesophilic 
                 
                   Wolbachia endosymbiont 
                 
               
               
                 304 
                 NC_020908|YP_007697819.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 + 
                 Mesophilic 
                 
                   Octadecabacter arcticus 
                 
               
               
                 305 
                 NC_017052|YP_005407905.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Mesophilic 
                   Synechocystis  sp. 
               
               
                 306 
                 NC_013532|YP_003328084.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Mesophilic 
                 
                   Anaplasma centrale 
                 
               
               
                 307 
                 NC_008599|YP_891325.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Mesophilic 
                 
                   Campylobacter fetus 
                 
               
               
                 308 
                 NC_023063|EMUR_00615 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Mesophilic 
                 
                   Ehrlichia muris 
                 
               
               
                 309 
                 NC_009952|YP_001533361.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Mesophilic 
                 
                   Dinoroseobacter shibae 
                 
               
               
                 310 
                 NC_011726|YP_002371657.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Mesophilic 
                   Cyanothece  sp. 
               
               
                 311 
                 NC_017038|YP_005382028.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Mesophilic 
                   Synechocystis  sp. 
               
               
                 312 
                 NC_021661|PSYCG_08920 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Mesophilic 
                   Psychrobacter  sp. 
               
               
                 313 
                 NC_014414|YP_003855886.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Mesophilic 
                 
                   Parvularcula bermudensis 
                 
               
               
                 314 
                 NC_008209|RD1_0916 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Mesophilic 
                 
                   Roseobacter denitrificans 
                 
               
               
                 315 
                 NC_007948|YP_549904.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Mesophilic 
                   Polaromonas  sp. 
               
               
                 316 
                 NC_007969|YP_580989.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Psychrophilic 
                 
                   Psychrobacter cryohalolentis 
                 
               
               
                 317 
                 NC_009659|YP_001353939.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Mesophilic 
                   Janthinobacterium  sp. 
               
               
                 318 
                 NC_013161|YP_003137220.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Mesophilic 
                   Cyanothece  sp. 
               
               
                 319 
                 NC_019729|YP_007113899.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 + 
                 Mesophilic 
                   Oscillatoria nigro - viridis   
               
               
                 320 
                 NC_022132|YP_008472938.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Mesophilic 
                 
                   Campylobacter coli 
                 
               
               
                 321 
                 NC_023018|X636_20130 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 + 
                 Mesophilic 
                   Pandoraea  sp. 
               
               
                 322 
                 NC_011726|YP_002371656.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Mesophilic 
                   Cyanothece  sp. 
               
               
                 323 
                 NC_022904|U875_14655 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 + 
                 Mesophilic 
                 
                   Pandoraea pnomenusa 
                 
               
               
                 324 
                 NC_015497|YP_004434941.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 + 
                 Mesophilic 
                   Glaciecola  sp. 
               
               
                 325 
                 NC_015559|YP_004480592.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Mesophilic 
                 
                   Marinomonas posidonica 
                 
               
               
                 326 
                 NC_020516|YP_007551100.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Mesophilic 
                   Azoarcus  sp. 
               
               
                 327 
                 NC_020417|YP_007501864.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 + 
                 Mesophilic 
                 beta proteobacterium 
               
               
                 328 
                 NC_021881|YYY_00245 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.39 
                 − 
                 Mesophilic 
                 
                   Anaplasma phagocytophilum 
                 
               
               
                 329 
                 NC_022529|YP_008623108.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Campylobacter jejuni 
                 
               
               
                 330 
                 NC_022362|YP_008561568.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Campylobacter jejuni 
                 
               
               
                 331 
                 NC_013161|YP_003137221.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                   Cyanothece  sp. 
               
               
                 332 
                 NC_019738|YP_007124477.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 + 
                 Mesophilic 
                   Microcoleus  sp. 
               
               
                 333 
                 NC_009839|YP_001481745.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Campylobacter jejuni 
                 
               
               
                 334 
                 NC_020911|YP_007706474.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 + 
                 Mesophilic 
                 
                   Octadecabacter antarcticus 
                 
               
               
                 335 
                 NC_019936|YP_007238420.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Pseudomonas stutzeri 
                 
               
               
                 336 
                 NC_016617|YP_005030415.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 + 
                 Mesophilic 
                 
                   Azospirillum brasilense 
                 
               
               
                 337 
                 NC_021879|YYU_00250 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Anaplasma phagocytophilum 
                 
               
               
                 338 
                 NC_021880|WSQ_00245 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Anaplasma phagocytophilum 
                 
               
               
                 339 
                 NC_014634|YP_003968941.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Ilyobacter polytropus 
                 
               
               
                 340 
                 NC_007298|YP_284282.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Dechloromonas aromatica 
                 
               
               
                 341 
                 NC_007797|YP_504684.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Anaplasma phagocytophilum 
                 
               
               
                 342 
                 NC_018709|YP_006857279.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Campylobacter jejuni 
                 
               
               
                 343 
                 NC_007798|YP_505908.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Neorickettsia sennetsu 
                 
               
               
                 344 
                 NC_002163|YP_002343633.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Campylobacter jejuni 
                 
               
               
                 345 
                 NC_013166|YP_003147637.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Kangiella koreensis 
                 
               
               
                 346 
                 NC_009925|YP_001514473.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Acaryochloris marina 
                 
               
               
                 347 
                 NC_011662|YP_002889566.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                   Thauera  sp. 
               
               
                 348 
                 NC_019683|YP_007069953.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 + 
                 Mesophilic 
                   Leptolyngbya  sp. 
               
               
                 349 
                 NC_021084|YP_007885545.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Wolbachia endosymbiont 
                 
               
               
                 350 
                 NC_015730|YP_004692622.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Roseobacter litoralis 
                 
               
               
                 351 
                 NC_015672|YP_004603167.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Thermophilic 
                 
                   Flexistipes sinusarabici 
                 
               
               
                 352 
                 NC_018267|YP_006555929.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Wolbachia endosymbiont 
                 
               
               
                 353 
                 NC_008340|YP_743404.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Alkalilimnicola ehrlichii 
                 
               
               
                 354 
                 NC_009524|YP_001279771.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                   Psychrobacter  sp. 
               
               
                 355 
                 NC_019567|YP_007024115.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 + 
                 Mesophilic 
                 
                   Bdellovibrio bacteriovorus 
                 
               
               
                 356 
                 NC_017506|YP_005887535.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 ? 
                 
                   Marinobacter adhaerens 
                 
               
               
                 357 
                 NC_019776|YP_007162494.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 + 
                 Mesophilic 
                 
                   Cyanobacterium aponinum 
                 
               
               
                 358 
                 NC_015740|YP_004716401.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Pseudomonas stutzeri 
                 
               
               
                 359 
                 NC_007802|YP_508060.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                   Jannaschia  sp. 
               
               
                 360 
                 NC_006576|YP_172630.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Synechococcus elongatus 
                 
               
               
                 361 
                 NC_006833|YP_198228.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Wolbachia endosymbiont 
                 
               
               
                 362 
                 NC_008789|YP_001002765.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.38 
                 − 
                 Mesophilic 
                 
                   Halorhodospira halophila 
                 
               
               
                 363 
                 NC_010475|YP_001735740.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Thermophilic 
                   Synechococcus  sp. 
               
               
                 364 
                 NC_009719|YP_001411459.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Parvibaculum lavamentivorans 
                 
               
               
                 365 
                 NC_013515|YP_003306318.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Streptobacillus moniliformis 
                 
               
               
                 366 
                 NC_014532|YP_003899070.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Halomonas elongata 
                 
               
               
                 367 
                 NC_007404|YP_314748.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Thiobacillus denitrificans 
                 
               
               
                 368 
                 NC_008228|YP_661172.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Pseudoalteromonas atlantica 
                 
               
               
                 369 
                 NC_009434|PST_4066 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Pseudomonas stutzeri 
                 
               
               
                 370 
                 NC_009654|YP_001342678.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                   Marinomonas  sp. 
               
               
                 371 
                 NC_022660|N149_1535 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Campylobacter coli 
                 
               
               
                 372 
                 NC_017280|YP_005657152.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Campylobacter jejuni 
                 
               
               
                 373 
                 NC_017279|YP_005655535.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Campylobacter jejuni 
                 
               
               
                 374 
                 NC_019695|YP_007092604.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 + 
                 Mesophilic 
                 
                   Chroococcidiopsis thermalis 
                 
               
               
                 375 
                 NC_022351|YP_008538079.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Campylobacter jejuni 
                 
               
               
                 376 
                 NC_022352|YP_008539796.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Campylobacter jejuni 
                 
               
               
                 377 
                 NC_019978|YP_007314869.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 + 
                 Mesophilic 
                 
                   Halobacteroides halobius 
                 
               
               
                 378 
                 NC_007614|YP_4U431.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Nitrosospira multiformis 
                 
               
               
                 379 
                 NC_008312|YP_722952.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Trichodesmium erythraeum 
                 
               
               
                 380 
                 NC_003912|YP_178192.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Campylobacter jejuni 
                 
               
               
                 381 
                 NC_018028|YP_006455879.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Pseudomonas stutzeri 
                 
               
               
                 382 
                 NC_017281|YP_005658777.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Campylobacter jejuni 
                 
               
               
                 383 
                 NC_022347|YP_008534235.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Campylobacter coli 
                 
               
               
                 384 
                 NC_013009|YP_003081241.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Neorickettsia risticii 
                 
               
               
                 385 
                 NC_022353|YP_008541533.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Campylobacter jejuni 
                 
               
               
                 386 
                 NC_019566|YP_007020209.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Candidatus Endolissoclinum 
                 
               
               
                 387 
                 NC_010524|YP_001791320.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Leptothrix cholodnii 
                 
               
               
                 388 
                 NC_015380|YP_004358590.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Candidatus Pelagibacter 
                 
               
               
                 389 
                 NC_021834|M635_05205 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Vibrio parahaemolyticus 
                 
               
               
                 390 
                 NC_019779|YP_007169582.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 + 
                 Mesophilic 
                   Halothece  sp. 
               
               
                 391 
                 NC_007604|YP_401192.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Synechococcus elongatus 
                 
               
               
                 392 
                 NC_009707|YP_001397430.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Campylobacter jejuni 
                 
               
               
                 393 
                 NC_008261|YP_694897.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 + 
                 Mesophilic 
                 
                   Clostridium perfringens 
                 
               
               
                 394 
                 NC_013177|YP_006526031.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Pseudomonas stutzeri 
                 
               
               
                 395 
                 NC_018521|YP_006632382.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Campylobacter jejuni 
                 
               
               
                 396 
                 NC_017532|YP_005940813.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.37 
                 − 
                 Mesophilic 
                 
                   Pseudomonas stutzeri 
                 
               
               
                 397 
                 NC_014147|YP_003626460.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Moraxella catarrhalis 
                 
               
               
                 398 
                 NC_007298|YP_284426.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Dechloromonas aromatica 
                 
               
               
                 399 
                 NC_003366|NP_561354.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 + 
                 Mesophilic 
                 
                   Clostridium perfringens 
                 
               
               
                 400 
                 NC_015410|YP_004378171.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Pseudomonas mendocina 
                 
               
               
                 401 
                 NC_015581|YP_004537902.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Thioalkalimicrobium cyclicum 
                 
               
               
                 402 
                 NC_007205|YP_266646.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Candidatus Pelagibacter 
                 
               
               
                 403 
                 NC_007963|YP_572610.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Chromohalobacter salexigens 
                 
               
               
                 404 
                 NC_008787|YP_999899.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Campylobacter jejuni 
                 
               
               
                 405 
                 NC_008347|YP_756198.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Maricaulis maris 
                 
               
               
                 406 
                 NC_012559|LHK_02634 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Laribacter hongkongensis 
                 
               
               
                 407 
                 NC_022360|M802.5393 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Pseudomonas aeruginosa 
                 
               
               
                 408 
                 NC_019701|YP_007102660.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 + 
                 Mesophilic 
                   Pseudanabaena  sp. 
               
               
                 409 
                 NC_019702|YP_007105817.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Thermophilic 
                   Synechococcus  sp. 
               
               
                 410 
                 NC_010546|YP_001804046.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                   Cyanothece  sp. 
               
               
                 411 
                 NC_022806|PA1R_gp3150 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Pseudomonas aeruginosa 
                 
               
               
                 412 
                 NC_015379|YP_004356944.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Pseudomonas brassicacearum 
                 
               
               
                 413 
                 NC_019905|YP_007231935.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Pseudomonas putida 
                 
               
               
                 414 
                 NC_020829|YP_007655716.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Pseudomonas denitrificans 
                 
               
               
                 415 
                 NC_013771|UCYN_05550 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Candidatas Atelocyanobacterium 
                 
               
               
                 416 
                 NC_018691|YP_006818918.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 + 
                 Mesophilic 
                 
                   Alcanivorax dieselolei 
                 
               
               
                 417 
                 NC_007604|YP_400426.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Synechococcus elongatus 
                 
               
               
                 418 
                 NC_018220|YP_006535054.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Pseudomonas putida 
                 
               
               
                 419 
                 NC_002516|PA5217 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Pseudomonas aeruginosa 
                 
               
               
                 420 
                 NC_006138|YP_065132.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Psychrophilic 
                 
                   Desulfotalea psychrophila 
                 
               
               
                 421 
                 NC_005085|CV1902 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Chromobacterium violaceum 
                 
               
               
                 422 
                 NC_006576|YP_170856.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Synechococcus elongatus 
                 
               
               
                 423 
                 NC_018080|YP_006485484.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Pseudomonas aeruginosa 
                 
               
               
                 424 
                 NC_009512|YP_001270405.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Pseudomonas putida 
                 
               
               
                 425 
                 NC_023076|X970_24575 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Pseudomonas monteilii 
                 
               
               
                 426 
                 NC_008463|YP_793689.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Pseudomonas aeruginosa 
                 
               
               
                 427 
                 NC_023066|T223_28685 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.36 
                 − 
                 Mesophilic 
                 
                   Pseudomonas aeruginosa 
                 
               
               
                 428 
                 NC_011770|YP_002443189.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas aeruginosa 
                 
               
               
                 429 
                 NC_022594|N297_5395 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas aeruginosa 
                 
               
               
                 430 
                 NC_017080|YP_005446529.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 + 
                 Mesophilic 
                 
                   Phycisphaera mikurensis 
                 
               
               
                 431 
                 NC_013194|YP_003167150.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Candidatas Accumulibacter 
                 
               
               
                 432 
                 NC_023019|U769_28730 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas aeruginosa 
                 
               
               
                 433 
                 NC_010501|YP_001751866.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas putida 
                 
               
               
                 434 
                 NC_020813|YP_007644977.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 + 
                 Mesophilic 
                 
                   Bdellovibrio exovorus 
                 
               
               
                 435 
                 NC_021577|M062_27505 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas aeruginosa 
                 
               
               
                 436 
                 NC_007577|YP_397756.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Prochlorococcus marinus 
                 
               
               
                 437 
                 NC_008260|YP_694317.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 + 
                 Mesophilic 
                 
                   Alcanivorax borkumensis 
                 
               
               
                 438 
                 NC_017548|YP_005977914.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas aeruginosa 
                 
               
               
                 439 
                 NC_017530|YP_005932744.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas putida 
                 
               
               
                 440 
                 NC_012560|YP_002801810.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Azotobacter vinelandii 
                 
               
               
                 441 
                 NC_011884|YP_002484191.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                   Cyanothece  sp. 
               
               
                 442 
                 NC_012660|YP_002875363.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas fluorescens 
                 
               
               
                 443 
                 NC_022738|PVLB_24670 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                   Pseudomonas  sp. 
               
               
                 444 
                 NC_019670|YP_007032226.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                   Pseudomonas  sp. 
               
               
                 445 
                 NC_019680|YP_007060009.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Thermophilic 
                   Synechococcus  sp. 
               
               
                 446 
                 NC_015222|YP_004294728.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                   Nitrosomonas  sp. 
               
               
                 447 
                 NC_021150|YP_007900667.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Azotobacter vinelandii 
                 
               
               
                 448 
                 NC_015733|YP_004704456.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas putida 
                 
               
               
                 449 
                 NC_017038|YP_005384390.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                   Synechocystis  sp. 
               
               
                 450 
                 NC_017039|YP_005387559.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                   Synechocystis  sp. 
               
               
                 451 
                 NC_021237|YP_008003151.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas protegens 
                 
               
               
                 452 
                 NC_021499|YP_008105776.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas resinovorans 
                 
               
               
                 453 
                 NC_016002|YP_004846419.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 + 
                 Mesophilic 
                   Pseudogulbenkiania  sp. 
               
               
                 454 
                 NC_017986|YP_006386336.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas putida 
                 
               
               
                 455 
                 NC_002947|NP_747297.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas putida 
                 
               
               
                 456 
                 NC_005773|YP_272537.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas syringae 
                 
               
               
                 457 
                 NC_004129|YP_263022.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas protegens 
                 
               
               
                 458 
                 NC_009656|YP_001351278.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas aeruginosa 
                 
               
               
                 459 
                 NC_009465|YP_001219493.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Candidatus Vesicomyosocius 
                 
               
               
                 460 
                 NC_023075|χ969_24940 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas monteilii 
                 
               
               
                 461 
                 NC_022591|N296_5395 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas aeruginosa 
                 
               
               
                 462 
                 NC_022361|M801_5260 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas aeruginosa 
                 
               
               
                 463 
                 NC_010322|YP_001671474.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas putida 
                 
               
               
                 464 
                 NC_022808|PA1S_gp3150 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas aeruginosa 
                 
               
               
                 465 
                 NC_020912|YP_007712322.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas aeruginosa 
                 
               
               
                 466 
                 NC_017052|YP_005410266.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                   Synechocystis  sp. 
               
               
                 467 
                 NC_021291|YP_008046709.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 + 
                 Mesophilic 
                 
                   Spiribacter salinus 
                 
               
               
                 468 
                 NC_021491|L483_31150 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas putida 
                 
               
               
                 469 
                 NC_016830|YP_005211024.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas fluorescens 
                 
               
               
                 470 
                 NC_014501|YP_003887996.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                   Cyanothece  sp. 
               
               
                 471 
                 NC_021505|YP_008116305.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas putida 
                 
               
               
                 472 
                 NC_008027|YP_610714.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas entomophila 
                 
               
               
                 473 
                 NC_000911|NP_442521.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                   Synechocystis  sp. 
               
               
                 474 
                 NC_004757|NP_841102.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Nitrosomonas europaea 
                 
               
               
                 475 
                 NC_017549|YP_005984172.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas aeruginosa 
                 
               
               
                 476 
                 NC_023149|SCV20265_5937 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.35 
                 − 
                 Mesophilic 
                 
                   Pseudomonas aeruginosa 
                 
               
               
                 477 
                 NC_010296|YP_001660682.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 − 
                 Mesophilic 
                 
                   Microcystis aeruginosa 
                 
               
               
                 478 
                 NC_021149|YP_007895619.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 − 
                 Mesophilic 
                 
                   Azotobacter vinelandii 
                 
               
               
                 479 
                 NC_015731|YP_004694426.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 − 
                 Mesophilic 
                   Nitrosomonas  sp. 
               
               
                 480 
                 NC_015556|YP_004472575.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 − 
                 Mesophilic 
                 
                   Pseudomonas fulva 
                 
               
               
                 481 
                 NC_004578|NP_790164.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 − 
                 Mesophilic 
                 
                   Pseudomonas syringae 
                 
               
               
                 482 
                 NC_008816|YP_001009743.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 − 
                 Mesophilic 
                 
                   Prochlorococcus marinus 
                 
               
               
                 483 
                 NC_008817|YP_001011645.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 − 
                 Mesophilic 
                 
                   Prochlorococcus marinus 
                 
               
               
                 484 
                 NC_022664|SPICUR_06490 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 + 
                 Mesophilic 
                   Spiribacter  sp. 
               
               
                 485 
                 NC_007492|YP_351158.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 − 
                 Mesophilic 
                 
                   Pseudomonas fluorescens 
                 
               
               
                 486 
                 NC_017911|YP_006326554.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 − 
                 Mesophilic 
                 
                   Pseudomonas fluorescens 
                 
               
               
                 487 
                 NC_018643|YP_006756812.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 + 
                 Mesophilic 
                 alpha proteobacterium 
               
               
                 488 
                 NC_009482|YP_001227890.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 − 
                 Thermophilic 
                   Synechococcus  sp. 
               
               
                 489 
                 NC_012032|Chy400_0349 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 − 
                 Mesophilic 
                   Chloroflexus  sp. 
               
               
                 490 
                 NC_007005|YP_233355.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 − 
                 Mesophilic 
                 
                   Pseudomonas syringae 
                 
               
               
                 491 
                 NC_004113|NP_681303.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 + 
                 Thermophilic 
                 
                   Thermosynechococcus elongatus 
                 
               
               
                 492 
                 NC_008319|YP_730750.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 − 
                 Thermophilic 
                   Synechococcus  sp. 
               
               
                 493 
                 NC_023064|U771_3037S 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 − 
                 Mesophilic 
                   Pseudomonas  sp. 
               
               
                 494 
                 NC_012881|YP_002993137.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 + 
                 Mesophilic 
                 
                   Desulfovibrio salexigens 
                 
               
               
                 495 
                 NC_023033|NK55_11575 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 + 
                 Mesophilic 
                   Thermosynechococcus  sp. 
               
               
                 496 
                 NC_019780|YP_007173026.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 + 
                 Mesophilic 
                 
                   Dactylococcopsis salina 
                 
               
               
                 497 
                 NC_005072|NP_893281.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 − 
                 Mesophilic 
                 
                   Prochlorococcus marinus 
                 
               
               
                 498 
                 NC_009482|YP_001227889.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 − 
                 Thermophilic 
                   Synechococcus  sp. 
               
               
                 499 
                 NC_008610|YP_903912.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 − 
                 Mesophilic 
                 
                   Candidatus Ruthia 
                 
               
               
                 500 
                 NC_011729|YP_002376116.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 − 
                 Mesophilic 
                   Cyanothece  sp. 
               
               
                 501 
                 NC_010175|Caur_0325 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 − 
                 Thermophilic 
                 
                   Chloroflexus aurantiacus 
                 
               
               
                 502 
                 NC_019689|YP_007080757.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 + 
                 Mesophilic 
                   Pleurocapsa  sp. 
               
               
                 503 
                 NC_020209|YP_007399509.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 − 
                 Mesophilic 
                 
                   Pseudomonas poae 
                 
               
               
                 504 
                 NC_019778|YP_007165071.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 + 
                 Mesophilic 
                 
                   Cyanobacterium stanieri 
                 
               
               
                 505 
                 NC_009840|YP_001484574.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 − 
                 Mesophilic 
                 
                   Prochlorococcus marinus 
                 
               
               
                 506 
                 NC_009976|YP_001551140.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 − 
                 Mesophilic 
                 
                   Prochlorococcus marinus 
                 
               
               
                 507 
                 NC_005070|NP_897888.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 − 
                 Thermophilic 
                   Synechococcus  sp. 
               
               
                 508 
                 NC_013173|YP_003157279.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 − 
                 Mesophilic 
                 
                   Desulfomicrobium baculatum 
                 
               
               
                 509 
                 NC_007516|YP_381882.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 − 
                 Thermophilic 
                   Synechococcus  sp. 
               
               
                 510 
                 NC_022579|YP_008685335.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 + 
                 Mesophilic 
                 
                   Desulfovibrio hydrothermalis 
                 
               
               
                 511 
                 NC_020055|YP_007326536.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 + 
                 Mesophilic 
                 
                   Desulfovibrio hydrothermalis 
                 
               
               
                 512 
                 NC_007498|YP_006716608.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 − 
                 Mesophilic 
                 
                   Pelobacter carbinolicus 
                 
               
               
                 513 
                 NC_008819|YP_001015440.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 − 
                 Mesophilic 
                 
                   Prochlorococcus marinus 
                 
               
               
                 514 
                 NC_009091|YP_001091585.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 − 
                 Mesophilic 
                 
                   Prochlorococcus marinus 
                 
               
               
                 515 
                 NC_007513|YP_378003.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 − 
                 Thermophilic 
                   Synechococcus  sp. 
               
               
                 516 
                 NC_007335|YP_291968.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 − 
                 Mesophilic 
                 
                   Prochlorococcus marinus 
                 
               
               
                 517 
                 NC_008820|YP_001018048.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 − 
                 Mesophilic 
                 
                   Prochlorococcus marinus 
                 
               
               
                 518 
                 NC_008358|YP_761373.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 − 
                 Mesophilic 
                 
                   Hyphomonas neptunium 
                 
               
               
                 519 
                 NC_019675|YP_007047725.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 + 
                 Mesophilic 
                 
                   Cyanobium gracile 
                 
               
               
                 520 
                 NC_005071|NP_894120.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.31 
                 − 
                 Mesophilic 
                 
                   Prochlorococcus marinus 
                 
               
               
                 521 
                 NC_009481|YP_001225531.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.3 
                 − 
                 Thermophilic 
                   Synechococcus  sp. 
               
               
                 522 
                 NC_005042|Pro1272 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.3 
                 − 
                 Mesophilic 
                 
                   Prochlorococcus marinus 
                 
               
               
                 523 
                 NC_014355|YP_003796723.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.27 
                 − 
                 Mesophilic 
                 
                   Candidatus Nitrospira 
                 
               
               
                   
               
            
           
         
       
     
     The ttFeBP5 sequence homologs contained 626 hits for which the PCS was off by one residue (H=1). Of these, 522 corresponded to all the hits predicted to be Fe III —HCO 3  binding proteins in the mhFeBP1 homolog family (i.e. H=0). Similarly, of the 142 hits identified in hmFeBP1 sequence homologs with one PCS residue missing (H=1), 70 were identified as H=0 sequences in the ttFeBP5 homolog family. These observations clearly supports that there are two subtly different schemes for coordinating Fe III , which can both be identified using a PCS filter with the appropriate positional information. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 ttFeBP5 homologs. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 # 
                 Name 
                 30 
                 120 
                 162 
                 219 
                 220 
                 Identity 
                 Gram 
                 Temperature 
                 Organism 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 4ELR| 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                   
                   
                   
                   
               
               
                 2 
                 NC_006461|YP_144894.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 1 
                 − 
                 Thermophilic 
                 
                   Thermus thermophilus 
                 
               
               
                 3 
                 NC_005835|YP_005233.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.99 
                 − 
                 Thermophilic 
                 
                   Thermus thermophilus 
                 
               
               
                 4 
                 NC_017587|YP_006058112.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.95 
                 − 
                 Thermophilic 
                 
                   Thermus thermophilus 
                 
               
               
                 5 
                 NC_017272|YP_005641170.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.93 
                 − 
                 Thermophilic 
                 
                   Thermus thermophilus 
                 
               
               
                 6 
                 NC_014974|YP_004203468.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.84 
                 − 
                 Thermophilic 
                 
                   Thermus scotoductus 
                 
               
               
                 7 
                 NC_017278|YP_005653498.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.83 
                 − 
                 Mesophilic 
                   Thermus  sp. 
               
               
                 8 
                 NC_019386|YP_006971576.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.82 
                 − 
                 Mesophilic 
                 
                   Thermus oshimai 
                 
               
               
                 9 
                 NC_013946|YP_003507791.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.67 
                 + 
                 Thermophilic 
                 
                   Meiothermus ruber 
                 
               
               
                 10 
                 NC_015387|YP_004366938.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.57 
                 − 
                 Thermophilic 
                 
                   Marinithermus hydrothermalis 
                 
               
               
                 11 
                 NC_019793|YP_007180090.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.55 
                 + 
                 Mesophilic 
                 
                   Deinococcus peraridilitoris 
                 
               
               
                 12 
                 NC_014212|YP_003686074.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.54 
                 + 
                 Thermophilic 
                 
                   Meiothermus silvanus 
                 
               
               
                 13 
                 NC_014032|YP_003572493.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.52 
                 − 
                 Mesophilic 
                 
                   Salinibacter ruber 
                 
               
               
                 14 
                 NC_007677|YP_446499.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.52 
                 − 
                 Mesophilic 
                 
                   Salinibacter ruber 
                 
               
               
                 15 
                 NC_015161|YP_004256093.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.5 
                 + 
                 Mesophilic 
                 
                   Deinococcus proteolyticus 
                 
               
               
                 16 
                 NC_012526|YP_002786654.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.49 
                 + 
                 Mesophilic 
                 
                   Deinococcus deserti 
                 
               
               
                 17 
                 NZ_AOLM00000000|WP_007274470.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 + 
                 Mesophilic 
                 
                   Haloferax sulfurifontis 
                 
               
               
                 18 
                 NZ_AOLP00000000|WP_004970546.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 + 
                 Mesophilic 
                 
                   Haloferax denitrificans 
                 
               
               
                 19 
                 NC_014729|YP_004036524.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.34 
                 − 
                 Mesophilic 
                 
                   Halogeometricum borinquense 
                 
               
               
                 20 
                 NC_013967|YP_003535749.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 + 
                 Mesophilic 
                 
                   Haloferax volcanii 
                 
               
               
                 21 
                 NZ_AOLL00000000|WP_006600212.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 + 
                 Mesophilic 
                 
                   Haloferax alexandrinus 
                 
               
               
                 22 
                 NZ_AOLI00000000|WP_004976562.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 + 
                 Mesophilic 
                 
                   Haloferax gibbonsii 
                 
               
               
                 23 
                 NZ_AOLH00000000|WP_004063130.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 + 
                 Mesophilic 
                 
                   Haloferax lucentense 
                 
               
               
                 24 
                 NZ_AOLE00000000|WP_008574569.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 + 
                 Mesophilic 
                   Haloferax  sp. 
               
               
                 25 
                 NC_017941|HFX_1795 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 + 
                 Mesophilic 
                 
                   Haloferax mediterranei 
                 
               
               
                 26 
                 NZ_AOLF00000000|WP_008574569.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 + 
                 Mesophilic 
                   Haloferax  sp. 
               
               
                 27 
                 NZ_AOLD00000000|WP_008574569.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 + 
                 Mesophilic 
                   Haloferax  sp. 
               
               
                 28 
                 NZ_ARPY01000000|WP_021054224.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 − 
                 Mesophilic 
                 
                   Haloquadratum walsbyi 
                 
               
               
                 29 
                 NZ_AOLG00000000|WP_008093701.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.33 
                 + 
                 Mesophilic 
                 
                   Haloferax prahovense 
                 
               
               
                 30 
                 NZ_AOLI00000000|WP_007540595.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 + 
                 Mesophilic 
                 
                   Haloferax larsenii 
                 
               
               
                 31 
                 NZ_AOLK00000000|WP_008324506.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 + 
                 Mesophilic 
                 
                   Haloferax elongans 
                 
               
               
                 32 
                 NZ_AOLN00000000|WP_008317080.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 + 
                 Mesophilic 
                 
                   Haloferax mucosum 
                 
               
               
                 33 
                 NC_021716|YP_008191219.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 − 
                 Mesophilic 
                 
                   Alteromonas macleodii 
                 
               
               
                 34 
                 NC_021710|YP_008170211.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 − 
                 Mesophilic 
                 
                   Alteromonas macleodii 
                 
               
               
                 35 
                 NC_021712|YP_008174294.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 − 
                 Mesophilic 
                 
                   Alteromonas macleodii 
                 
               
               
                 36 
                 NC_021717|YP_008194574.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 − 
                 Mesophilic 
                 
                   Alteromonas macleodii 
                 
               
               
                 37 
                 NC_023045|I533_00730 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 − 
                 Mesophilic 
                 
                   Alteromonas macleodii 
                 
               
               
                 38 
                 NC_021713|YP_008178099.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 − 
                 Mesophilic 
                 
                   Alteromonas macleodii 
                 
               
               
                 39 
                 NC_021714|YP_008182148.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 − 
                 Mesophilic 
                 
                   Alteromonas macleodii 
                 
               
               
                 40 
                 NC_019393|YP_006975038.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 − 
                 Mesophilic 
                 
                   Alteromonas macleodii 
                 
               
               
                 41 
                 NC_011138|MADE_1000805 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 − 
                 Mesophilic 
                 
                   Alteromonas macleodii 
                 
               
               
                 42 
                 NC_008212|YP_658265.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 − 
                 Mesophilic 
                 
                   Haloquadratum walsbyi 
                 
               
               
                 43 
                 NC_018679|YP_006801087.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 − 
                 Mesophilic 
                 
                   Alteromonas macleodii 
                 
               
               
                 44 
                 NC_018692|YP_006823164.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.32 
                 − 
                 Mesophilic 
                 
                   Alteromonas macleodii 
                 
               
               
                 45 
                 NC_018632|YP_006746238.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.31 
                 − 
                 Mesophilic 
                 
                   Alteromonas macleodii 
                 
               
               
                 46 
                 NC_018678|YP_006797203.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.31 
                 − 
                 Mesophilic 
                 
                   Alteromonas macleodii 
                 
               
               
                 47 
                 NZ_AOLZ00000000|WP_007140174.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.31 
                 + 
                 Mesophilic 
                 
                   Halobiforma lacisaisi 
                 
               
               
                 48 
                 NC_017857|YP_006294772.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.31 
                 + 
                 Mesophilic 
                   Methylophaga  nitratireducentic 
               
               
                 49 
                 NC_015554|YP_004468950.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.31 
                 − 
                 Mesophilic 
                   Alteromonas  sp. 
               
               
                 50 
                 NZ_AOID00000000|WP_006430814.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.31 
                 + 
                 Mesophilic 
                 
                   Natrinema versiforme 
                 
               
               
                 51 
                 NC_020388|YP_007488351.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.31 
                 + 
                 Mesophilic 
                 
                   Natronomonas moolapensis 
                 
               
               
                 52 
                 NC_014297|YP_003737103.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.3 
                 N/a 
                 Mesophilic 
                 
                   Halalkalicoccus jeotgali 
                 
               
               
                 53 
                 NZ_AOJH00000000|WP_008848743.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.3 
                 + 
                 Mesophilic 
                 
                   Halorubrum kocurii 
                 
               
               
                 54 
                 NZ_AOJK00000000|WP_008444444.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.3 
                 + 
                 Mesophilic 
                 
                   Halorubrum californiensis 
                 
               
               
                 55 
                 NZ_AOJ100000000|WP_006111552.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.3 
                 + 
                 Mesophilic 
                 
                   Halorubrum coriense 
                 
               
               
                 56 
                 NC_021313|YP_008054586.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.3 
                 + 
                 Mesophilic 
                   Salinarchaeum  sp. 
               
               
                 57 
                 NC_007481|ΥP_340128.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.3 
                 − 
                 Psychrophilic 
                 
                   Pseudoalteromonas haloplanktis 
                 
               
               
                 58 
                 NZ_AOJF00000000|WP_004598669.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.3 
                 + 
                 Mesophilic 
                 
                   Halorubrum litoreum 
                 
               
               
                 59 
                 NZ_AOIW00000000|WP_004598669.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.3 
                 + 
                 Mesophilic 
                 
                   Halorubrum terrestre 
                 
               
               
                 60 
                 NZ_AOJN00000000|WP_004598669.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.3 
                 + 
                 Mesophilic 
                 
                   Halorubrum distributum 
                 
               
               
                 61 
                 NZ_AOJJ00000000|WP_004598669.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.3 
                 + 
                 Mesophilic 
                 
                   Halorubrum arcis 
                 
               
               
                 62 
                 NC_014803|YP_004068662.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.3 
                 − 
                 Psychrophilic 
                   Pseudoalteromonas  sp. 
               
               
                 63 
                 NZ_AOJI00000000|WP_008002218.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.29 
                 + 
                 Mesophilic 
                 
                   Halorubrum aidingense 
                 
               
               
                 64 
                 NZ_AGCY01000000|WP_021043411.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.29 
                 + 
                 Mesophilic 
                 
                   halophilic archaeon 
                 
               
               
                 65 
                 NC_012029|YP_002564837.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.29 
                 + 
                 Mesophilic 
                 
                   Halorubrum lacusprofundi 
                 
               
               
                 66 
                 NZ_AOJD00000000|WP_006629340.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.29 
                 + 
                 Mesophilic 
                 
                   Halorubrum tebenquichense 
                 
               
               
                 67 
                 NZ_AOIQ00000000|WP_007702269.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.29 
                 + 
                 Mesophilic 
                 
                   Halovivax asiaticus 
                 
               
               
                 68 
                 NC_019964|YP_007285378.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.29 
                 + 
                 Mesophilic 
                 
                   Halovivax ruber 
                 
               
               
                 69 
                 NZ_AOJG00000000|WP_008005399.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.29 
                 + 
                 Mesophilic 
                 
                   Halorubrum lipolyticum 
                 
               
               
                 70 
                 NZ_AOJO00000000|WP_008582685.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.29 
                 + 
                 Mesophilic 
                 
                   Halorubrum hochstenium 
                 
               
               
                 71 
                 NZ_AOIA00000000|WP_008420339.1 
                 R 
                 R 
                 Y 
                 Y 
                 Y 
                 0.29 
                 + 
                 Mesophilic 
                 
                   Natronococcus jeotgali 
                 
               
               
                   
               
            
           
         
       
     
     Example 3. Sensor Engineering Phase 2: Lead Protein Validation Using Ligand-Mediated Thermostability Shifts 
     The ligand-binding properties of the proteins selected were determined experimentally. These experiments comprise four successive steps:
         1. Synthetic gene construction. The amino acid sequence of the homology leads are backtranslated into DNA sequences. These are optimized for directing heterologous cytoplasmic expression of the protein homologues in  E. coli , using either the OrfOpt or OrfMorph programs. These programs predict mRNA sequences that direct high-level protein expression in  E. coli . The predicted gene sequences are assembled de novo from synthetic oligonucleotides.   2. Heterologous protein expression of the homologues in  E. coli . Plasmids carrying the synthetic expression constructs (see above) were transformed into KRX  E. coli  K12 derivative strain) competent cells (Promega, Technical Bulletin TB352). Protein production was induced in bacterial cultures of these cultures, as described in the Materials and Methods.   3. Purification of successfully expressed protein using immobilized metal affinity chromatography, as described in Materials and Methods.   4. Verification of ligand binding. Determination of the ligand-binding properties of the purified proteins using a thermal stability shift assay, as described in Materials and Methods.       

     Lead Verification by Thermal Shift Analysis of Bicarbonate-Calcium Binding Proteins. 
     The lead sequence and five additional homologs with identical PCS sequences (i.e. H=0) were selected, probing different degrees of sequence identity with the seed ( FIG. 5 ). Synthetic genes with open reading frames optimized for heterologous protein over-expression in  Escherichia coli  using OrfOpt (Allert, Cox and Hellinga 2010) were synthesized for each lead. Three of the six leads produced soluble protein in a T7 expression system in sufficient quantity for functional analysis. The proteins were purified by immobilized metal affinity chromatography via a hexa-histidine tag fused to their carboxy-terminus. Bicarbonate binding was tested in a thermal stability shift assay (Layton and Hellinga 2010). All leads bound bicarbonate, and not nitrate (Table 4). The bicarbonate-binding protein from  Anabaena variabilis  (avBicarbBP5) was selected for further engineering. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Ligand-binding and thermostability properties of synBicarbBP homologs. 
               
            
           
           
               
               
               
            
               
                   
                 Thermo- 
                 Ligand 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 NCBI Accession codes 
                   
                 Soluble 
                 stability c   
                 binding d   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Name 
                 Organism 
                 Genome 
                 Protein 
                 Identity a   
                 Expression b   
                   apo T m  (C.) 
                 Bicarbonate 
                 Nitrate 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 synBicarbBP1 
                   Synechocystis  sp. 
                 NC_017052 
                 YP_005410477.1 
                 1 
                 n 
                   
                   
                   
               
               
                 teBicarbBP2 
                 
                   Thermosynechococcus elongatus 
                 
                 NC_004113 
                 NP_682790.1 
                 0.83 
                 n 
               
               
                 ctBicarbBP3 
                 
                   Chroococcidiopsis thermalis 
                 
                 NC_019695 
                 YP_007090308.1 
                 0.67 
                 y 
                 86 
                 y 
                 n 
               
               
                 calBicarbBP4 
                   Calothrix  sp. 
                 NC_019751 
                 YP_007137061.1 
                 0.65 
                 y 
                 89 
                 y 
                 n 
               
               
                 avBicarbBP5 
                 
                   Anabaena variabilis 
                 
                 NC_007413 
                 YP_321546.1 
                 0.66 
                 y 
                 82 
                 y 
                 n 
               
               
                 cmBicarbBP6 
                 
                   Chamaesiphon minutus 
                 
                 NC_019697 
                 YP_007099445.1 
                 0.63 
                 n 
               
               
                   
               
               
                   a Number of identical residues shared with the probe sequence. 
               
               
                   b Judged by SDS gel electrophoresis of the soluble fraction of a total lysate. 
               
               
                   c Determined in a Roche LightCycler, using SYPRO Orange to monitor the appearance of unfolded protein. 
               
               
                   d Determined by monitoring an increase in the thermostability of the protein in the presence of ligand. 
               
            
           
         
       
     
     Lead Verification by Thermal Shift Analysis of Ferric-Bicarbonate Binding Proteins. 
     For each of the two leads, the lead and three sequence homologs with identical PCS sequences were selected ( FIG. 6 ). Synthetic genes with open reading frames optimized for heterologous protein over-expression in  Escherichia coli  using OrfMorph (see Materials and Methods) were synthesized for each lead. Six of the eight leads produced soluble protein in a T7 expression system in sufficient quantity for functional analysis ( FIG. 6 ). The two leads from the ttFeBP5-seeded search precipitated upon storage. The proteins were purified by immobilized metal affinity chromatography via a hexa-histidine tag fused to their carboxy-terminus. Bicarbonate binding was tested in a thermal stability shift assay (Layton and Hellinga 2010). All four soluble leads bound bicarbonate (Table 5). The bicarbonate-binding protein from  Thermosynechococcus elongatus  (teFeBP3) was selected for further engineering. It is noteworthy that function was predicted accurately for homologs of considerably less than 60% sequence identity (the lowest is 26%), which is considered the lower limit for predicted function conservation on overall homology alone (Todd 2001, Tian 2003). The PCS-based method therefore is a powerful for the prediction of functional conservation in distantly related proteins. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Ligand-binding and thermostability properties of FeBP homologs. 
               
            
           
           
               
               
               
            
               
                   
                 Thermo- 
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 NCBI Accession codes 
                   
                 Soluble 
                 stability d   
                 Bicarbonate 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Probe a   
                 Name 
                 Organism 
                 Genome 
                 Protein 
                 Identity b   
                 Expression c   
                   apo T m  (C) 
                 binding e   
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 lsiO 
                 mhFeBP1 
                 
                   Mannheimia haemolytica 
                 
                 NC_0121082 
                 YP_007884192.1 
                 1 
                 y 
                 ~50 f   
                 y 
               
               
                   
                 exiFeBP2 
                   Exiguobacterium  sp. 
                 NC_012673 
                 YP_002886303.1 
                 0.41 
                 y 
                     37 
                 y 
               
               
                   
                 teFeBP3 
                 
                   Thermosynechoccus elongatus 
                 
                 NC_004113 
                 NP_681303.1 
                 0.34 
                 y 
                     67 
                 y 
               
               
                   
                 cnFeBP4 
                 
                   Candidatus nitrospira 
                 
                 NC_014355 
                 YP_003796723.1 
                 0.26 
                 y 
                 ~50 g   
                     y g   
               
               
                 4elr 
                 ttFeBP5 
                 
                   Thermus thermophilus 
                 
                 NC_006461 
                 YP_144894.1 
                 1 
                     y h   
               
               
                   
                 msFeBP6 
                 
                   Meiothermus silvanus 
                 
                 NC_014212 
                 YP_003686074.1 
                 0.54 
                     y h   
               
               
                   
                 srFeBP7 
                 
                   Salinibacter ruber 
                 
                 NC_014032 
                 YP_003572493.1 
                 0.52 
                 n 
               
               
                   
                 hlFeBP8 
                 
                   Halorubrum lacusprofundi 
                 
                 NC_012029 
                 YP_002564837.1 
                 0.31 
                 n 
               
               
                   
               
               
                   a PDB accession of the probe sequence and PCS definition (see Table 2-3). 
               
               
                   b Number of identical residues shared with the probe sequence. 
               
               
                   c Judged by SDS gel electrophoresis of the soluble fraction of a total lysate. 
               
               
                   d Determined in a Roche LightCycler, using SYPRO Orange to monitor the appearance of unfolded protein. 
               
               
                   e Determined by monitoring an increase in the thermostability of the protein in the presence of ligand. 
               
               
                   f Estimate: multi-state unfolding. 
               
               
                   g Estimated from incomplete data 
               
               
                   h Precipitated during dialysis. 
               
            
           
         
       
     
     Example 4. Sensor Engineering Phase 3: Cysteine Mutant Scans and Fluorophore Screening to Identify Fluorescently Responsive Sensors for Target Ligand 
     Semi-synthetic FRSs can be engineered by site-specifically attaching thiol-reactive, environmentally sensitive fluorophores that respond to ligand-mediated conformational changes. Identification of FRS candidates that can be used for sensing applications comprises three steps:
         1. Cysteine scan. Mutant ligand-binding proteins containing single cysteines are constructed for site-specific attachment of thiol-reactive fluorophores. General structural principles have been established to identify positions in PBPs where attached single fluorophores are likely to exhibit ligand-dependent responses (de Lorimier et al. 2002). Candidate positions fall into three classes: endosteric, replacing a residue that contacts the ligand directly; peristeric, located at the rim of the binding site; allosteric (Marvin et al. 1997. Marvin 1998), located outside the binding site at sites that undergo local structural changes in concert with the hinge-bending motion.   2. Fluorophore screening. Thiol-reactive, environmentally sensitive fluorophores are attached to each cysteine mutant prepared in step 1.   3. Evaluation of the ligand-mediated change of all the fluorescent conjugates prepared in step 2. Responses to ligand binding in which there is both a change in fluorescence emission intensity and spectral shape are essential for chemometric applications, because such changes enable ratiometric measurements. Changes in spectral shape typically are accompanied by a shift in the wavelength of the emission intensity maxima. Three classes of fluorescent responses are possible:
           i. No response.   ii. Monochromatic response (emission intensity increases or decreases without a change in spectral shape)   iii. Dichromatic response (both intensity and spectral shape changes) which can be classified into two sub-classes:
               i. Hypsochromatic: emission intensity shifts to shorter wavelengths upon binding ligand (“blue shift”).   ii. Bathochromatic: emission intensity shifts to longer wavelengths upon binding ligand (“red shift”).   
               
           4. Double labeling strategies to convert monochromatic responses into dichromatic signals, or to improve upon weak dichromatic responses.       

     Of particular interest are ligand-mediated responses that change both the shape and intensity of the emission spectra such that the ratio of the emission intensities at two appropriately chosen wavelengths reports on analyte concentration (dichromatic response). 
     Such ratiometric measurements provide an internally consistent, self-calibrating reference, which removes the necessity for carrying out on-board calibration in conjunction with each measurement, obviating the need for multiple components and fluidic circuitry (Demchenko 2010, Demchenko 2014). The naphthalene derivatives Acrylodan and Badan have been particularly effective in establishing dichromatic responses in singly labeled proteins, because ligand-mediated protein conformational changes can be coupled to an exchange between two different fluorophore conformations (twists) that emit at different wavelengths. Initial screens therefore employ these fluorophores to identify locations where fluorescent conjugates respond to ligand binding. Two dominant electronic transitions give rise to fluorescence emission in these fluorophores with maxima in the blue (&lt;500 nm) and green (&gt;500 nm) regions, respectively. 
     Cysteine Scans to Identify Semisynthetic Fluorescent Bicarbonate Sensors in avBicarbBP5. 
     We tested the bicarbonate responses of Acrylodan conjugates attached to cysteine mutations at peristeric and endosteric positions of avBicarbBP ( FIG. 3 ). With the exception of N146C. all positions exhibited monochromatic or no responses (Table 6). In the case of 146C, there was only a small shift in emission wavelengths, and no significant change in intensity. Several conjugates were predominantly in the green state (18C, 49C, 71C), or blue (141C, 190C) states, whereas others had a mixture of the two forms (16C, 17C, 140C, 143C, 146C, 194C). Acrylodan therefore could adopt both states in avBicarbBP5, but these did not alter in response to bicarbonate binding, with the exception of 146C. These results suggest that ligand binding is not accompanied by a large conformational change that couples to a redistribution of internal conjugate conformations. The structure of the hinge region in avBicarbBP5 clearly is not flexible, but resembles a small protein domain with a significant hydrophobic core ( FIG. 3 ). The observed absence of conformational coupling between bicarbonate binding and Acrylodan twisting therefore is consistent with the avBicarbBP5. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Bicarbonate response of Acrylodan conjugates in 
               
               
                 a cysteine scan of the avBicarbBPS scaffold. 
               
            
           
           
               
               
               
            
               
                   
                 Response 
                 Thermo- 
               
            
           
           
               
               
               
               
            
               
                 Position a   
                   
                 Excited 
                 stability f   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 avBicarbBP6 
                 synBicarbBP1 
                 Class b   
                 Shape c   
                 Intensity d   
                 states e   
                   apo T m  (K) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 I16C 
                 66 
                 p 
                 0 
                   
                 b/g 
                 334 
               
               
                 P17C 
                 67 
                 p 
                 m 
                 + 
                 b/g 
                 326 
               
               
                 I18C 
                 68 
                 e 
                 0 
                   
                 g 
                 336 
               
               
                 W49C 
                 99 
                 e 
                 0 
                   
                 g 
                 322 
               
               
                 Q71C 
                 121 
                 e 
                 0 
                   
                 g 
                 343 
               
               
                 F140C 
                 H191 
                 p 
                 m 
                 + 
                 b/g 
                 338 
               
               
                 T141C 
                 192 
                 e 
                 m 
                 + 
                 b 
                 331 
               
               
                 F142C 
                 193 
                 p 
                 m 
                 + 
                 g 
                 331 
               
               
                 P143C 
                 194 
                 p 
                 0 
                   
                 b/g 
                 325 
               
               
                 N146C 
                 197 
                 p 
                 d 
                 0 
                 b/g 
                 333 
               
               
                 T190C 
                 241 
                 p 
                 m 
                 + 
                 b 
                 329 
               
               
                 W194C 
                 245 
                 p 
                 m 
                 + 
                 b/g 
                 329 
               
               
                   
               
               
                   a Aligned position in the synBicarbBP1 numbering of the 2i4c PDB file is given. 
               
               
                   b e, endosteric; p, peristeric. 
               
               
                   c m, monochromatic; d, dichromatic (i.e. spectral shape changes); 0, no change. 
               
               
                   d +, increases in response to bicarbonate; decreases; 0, no change. 
               
               
                   e The dominant population of the excited states in the absence of bicarbonate is determined from the emission band intensities: b, blue (maxima &lt; 500 nm); g, green (maxima &gt; 500 nm); b/g, mixed population of blue and green. 
               
               
                   f Determined by thermal melts based on Acrylodan fluorescence. The wild-type protein  apo T m  = 355 K. 
               
            
           
         
       
     
     In the absence of conformational coupling between ligand binding and fluorophore twisting as a mechanism to obtain fluorescently responsive sensors, we tested Pacific Blue conjugates. We found that at positions 18C, 190C and 194C, Pacific Blue exhibited monochromatic changes in emission intensity in response to bicarbonate binding. Pacific Blue is a hydroxycoumarin derivative, the phenolate of which corresponds to the excited state (Sun 1998). It is therefore likely that binding of Ca 2+  stabilizes the excited state, with a concomitant increase in quantum yield. 
     Introducing Ratiometric Bicarbonate Responses in avBicarbBP5 Using Non-Geometrically Modulated FRET. 
     We have shown previously that fluorescence resonance energy transfer can be used to report on binding events by pairing an environmentally sensitive, ligand-responsive donor or acceptor with an insensitive partner. Ratiometric measurements are based on changes in donor and acceptor intensities in such non-geometric modulated FRET (ngmFRET) systems. The dichromatic response arises from ligand-mediated effects on the spectral overlap between the partners or the non-radiative decay rates of the sensitive fluorophore, either singly or in combination. 
     To convert the monochromatic Pacific Blue responses of the 18C and 194C conjugates into dichromatic signals, we constructed an ngmFRET system by fusing a C-terminal βZif domain which enables site-specific attachment a second fluorophore using orthogonal thiol chemistries (Smith et al. 2005). This avBicarbBP5 fusion protein was doubly labelled with Pacific Blue ngmFRET donor in the binding site and IANBD or the Fluorescein derivatives 5-IAF and Oregon Green as the ngmFRET acceptor at the C-terminal βZif. These conjugates exhibited strong dichromatic responses to bicarbonate binding ( FIG. 7 ). Both donor and acceptor emissions increased in response to bicarbonate binding, which is the expected ngmFRET pattern for ligand-mediated changes in the non-radiative decay rates of a responsive donor (Allert 2015). 
     Cysteine Scans to Identify Semisynthetic Fluorescent Bicarbonate Sensors in teFeBP3. 
     Several Acrylodan and Badan conjugates exhibited dichromatic responses to bicarbonate binding (Table 7). It is therefore possible to establish coupling between ligand-mediated protein conformational changes and conjugate twisting. The largest response was observed for the teFeBP3 E270C*Badan conjugate ( FIG. 8 ). The apparent affinity for bicarbonate, based on the ratiometric signal, is 48 mM, which is suitable for measurements in the clinical reference concentration range. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Bicarbonate response of Acrylodan conjugates in a cysteine 
               
               
                 scan of peristeric sites in the teFeBP3 scaffold. 
               
            
           
           
               
               
            
               
                   
                 Response 
               
            
           
           
               
               
               
            
               
                   
                 Acrylodan 
                 Badan 
               
            
           
           
               
               
               
               
               
            
               
                 Position a   
                   
                 Excited 
                 Inten- 
                 Excited 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 teFeBP3 
                 mhFeBP1 
                 Shape b   
                 Intensity c   
                 states d   
                 Shape b   
                 sity c   
                 states d   
               
               
                   
               
               
                 A8C 
                 Y9 
                 0 
                   
                 b/g 
                 m 
                 + 
                 b/g 
               
               
                 H10C 
                 Q11 
                 0 
                   
                 b/g 
                 d 
                 − 
                 b/g 
               
               
                 D12C 
                 Y13 
                 0 
                   
                 b/g 
                 0 
                   
                 g 
               
               
                 T13C 
                 L14 
                 0 
                   
                 b/g 
                 d 
                 − 
                 g 
               
               
                 A36C 
                  36 
                 m 
                 − 
                 b/g 
                 d 
                 − 
                 b 
               
               
                 V58C 
                  58 
                 d 
                 + 
                 b/g 
                 d 
                 + 
                 b/g 
               
               
                 R135C 
                 136 
                 m 
                 + 
                 g 
                 0 
                   
                 g 
               
               
                 N139C 
                 140 
                 m 
                 − 
                 g 
                 0 
                   
                 b 
               
               
                 I140C 
                 S141 
                 0 
                   
                 g 
                 m 
                 − 
                 g 
               
               
                 N176C 
                 G177 
                 0 
                   
                 g 
                 m 
                 − 
                 g 
               
               
                 N195C 
                 196 
                 0 
                   
                 g 
                 0 
                   
                 b 
               
               
                 N268C 
                 266 
                 0 
                   
                 b 
                 m 
                 − 
                 b/g 
               
               
                 E270C 
                 268 
                 d 
                 − 
                 g 
                 d 
                 − 
                 g 
               
               
                   
               
               
                   a Aligned position in the mhFeBP1 numbering of the 1si0 PDB file is given. 
               
               
                   b m, monochromatic; d, dichromatic (i.e. spectral shape changes); 0, no change. 
               
               
                   c +, increases in response to bicarbonate; decreases; 0, no change. In the presence of FeCl 3  (10 μM). 
               
               
                   d The dominant population of the excited states in the absence of bicarbonate is determined from the emission band intensities: b, blue (maxima &lt; 500 nm); g, green (maxima &gt; 500 nm); b/g, mixed population of blue and green. 
               
            
           
         
       
     
     Semisynthetic Fluorescent Calcium Sensors in avBicarbBP5. 
     We tested fluorescent conjugates for response to Ca 2+  in the absence of bicarbonate (Table 8). Pacific Blue conjugates exhibited excellent responses at two sites (16C and 18C) and showed responses at 17C, 49C, 140C and 142C. Badan exhibited dichromatic response to Ca 2+  at 17C, 18C, 140C, 142C and 194C ( FIG. 9 ). The 17C and 18C conjugates bind Ca 2+  with 1-2 mM affinities. Accordingly, these conjugates are well-suited for measuring Ca 2+  concentrations in the clinical reference range. 
     The cysteine scans also demonstrate that Ca 2+  and bicarbonate binding functions can be separated. The Ca 2+  affinities of the five 16C, 17C, 18C, 140C, and 142C conjugates change little, if at all, in the presence of 50 mM bicarbonate (Table 9). The avBicarbBP5 protein therefore can be engineered to function as either a Ca II —HCO 3  or a Ca 2+  sensor. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Calcium response in a cysteine scan of the avBicarbBP5 scaffold a . 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Emission 
                 K d   
               
               
                 Position b   
                 Class 
                 Conjugate 
                 Shape 
                 wavelength (nm) 
                 (mM) 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 avBicarbBP5 
                 synBicarbBP1 
                 
                   c 
                 
                 
                   d 
                 
                 
                   e 
                 
                 λ 1   
                 λ 2   
                   app K d   
                   true K d   
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 I16C 
                 66 
                 p 
                 A 
                 m 
                 481 
                 526 
                 22 
                 22 
               
               
                   
                   
                   
                 B 
                 0 
               
               
                   
                   
                   
                 PB 
                 m/d 
                 445 
                 475 
                 0.30 
                 0.64 
               
               
                   
                   
                   
                 A532 
                 m/d 
                 540 
                 582 
                 0.30 
                 0.71 
               
               
                 P17C 
                 67 
                 p 
                 A 
                 d 
                 443 
                 548 
                 1.9 
                 2.5 
               
               
                   
                   
                   
                 B 
                 d 
                 488 
                 553 
                 2.0 
                 2.4 
               
               
                   
                   
                   
                 PB 
                 m/d 
                 436 
                 479 
                 3.1 
                 14 
               
               
                   
                   
                   
                 A532 
                 nt 
                 555 
                 540 
                 24 
                 68 
               
               
                 I18C 
                 68 
                 e 
                 A 
                 d 
                 461 
                 544 
                 2.0 
                 2.7 
               
               
                   
                   
                   
                 B 
                 d 
                 441 
                 548 
                 1.1 f   
                 2.2 f   
               
               
                   
                   
                   
                 PB 
                 m/d 
                 444 
                 479 
                 2.7 
                 5.3 
               
               
                   
                   
                   
                 A532 
                 m 
                 559 
                 540 
                 2.4 
                 3.2 
               
               
                 W49C 
                 99 
                 e 
                 A 
                 0 
               
               
                   
                   
                   
                 B 
                 0 
               
               
                   
                   
                   
                 PB 
                 m 
                 442 
                 477 
                 41 
                 33 
               
               
                 Q71C 
                 121 
                 e 
                 A 
                 0 
               
               
                   
                   
                   
                 B 
                 0 
               
               
                   
                   
                   
                 PB 
                 0 
               
               
                 F140C 
                 H191 
                 p 
                 A 
                 d 
                 483 
                 520 
                 0.35 
                 0.41 
               
               
                   
                   
                   
                 B 
                 d 
                 440 
                 552 
                 0.19 
                 0.22 
               
               
                   
                   
                   
                 PB 
                 m 
                 451 
                 481 
                 0.28 
                 0.34 
               
               
                   
                   
                   
                 A532 
                 m 
                 549 
                 579 
                 2.0 
                 2.5 
               
               
                 T141C 
                 192 
                 e 
                 A 
                 d 
                 440 
                 531 
                 0.46 
                 0.53 
               
               
                   
                   
                   
                 B 
                 0 
               
               
                   
                   
                   
                 PB 
                 0 
               
               
                   
                   
                   
                 A532 
                 m/d 
                 555 
                 540 
                 0.6 
                 0.7 
               
               
                 F142C 
                 193 
                 p 
                 A 
                 d 
                 487 
                 518 
                 0.23 
                 0.22 
               
               
                   
                   
                   
                 B 
                 d 
                 483 
                 515 
                 0.2 
                 0.2 
               
               
                   
                   
                   
                 PB 
                 m 
                 460 
                 435 
                 0.04 
                 0.04 
               
               
                   
                   
                   
                 A532 
                 m/d 
                 555 
                 540 
                 0.31 
                 0.68 
               
               
                 P143C 
                 194 
                 p 
                 A 
                 m 
                 462 
                 522 
                 0.43 
                 0.42 
               
               
                   
                   
                   
                 B 
                 m 
                 488 
                 568 
                 0.92 
                 0.98 
               
               
                   
                   
                   
                 PB 
                 0 
               
               
                 N146C 
                 197 
                 p 
                 A 
                 0 
               
               
                   
                   
                   
                 B 
                 0 
               
               
                   
                   
                   
                 PB 
                 0 
               
               
                 T190C 
                 241 
                 p 
                 A 
                 d 
                 491 
                 451 
                 0.54 
                 0.64 
               
               
                   
                   
                   
                 B 
                 0 
               
               
                   
                   
                   
                 PB 
                 0 
               
               
                 W194C 
                 245 
                 p 
                 A 
                 d 
                 483 
                 510 
                 20 f   
                 10 f   
               
               
                   
                   
                   
                 B 
                 m/d 
                 487 
                 515 
                 1.2 
                 0.94 
               
               
                   
                   
                   
                 PB 
                 0 
               
               
                   
               
               
                   a Determined by fitting the ratiometric signal of the intensities measured at λ1 and λ2 to equations 1-5. 
               
               
                   b Aligned position in the synBicarbBP numbering of the 2i4c PDB file is given. 
               
               
                   c  e, endosteric; p, peristeric. 
               
               
                   d  A, Acrylodan; B, Badan; PB, Pacific Blue, A532, Alexa532. 
               
               
                   e  m, monochromatic; d, dichromatic (i.e. spectral shape changes); 0, no or very small change. 
               
               
                   f Approximate value. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Calcium affinities for various avBicarbBP5 conjugates 
               
               
                 in the presence and absence of NaHCO 3   a . 
               
            
           
           
               
               
            
               
                   
                 Affinity (mM) 
               
            
           
           
               
               
               
               
            
               
                   
                 Wavelengths 
                   
                 Calcium 
               
               
                   
                 (nm) 
                 Calcium 
                 (NaHCO 3 ) c   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Mutation 
                 Conjugate b   
                 λ 1   
                 λ 2   
                   app K d   
                   true K d   
                   app K d   
                   true K d   
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 16C 
                 PB 
                 445 
                 475 
                 0.30 
                 0.64 
                 0.27 
                 0.64 
               
               
                 17C 
                 B 
                 488 
                 553 
                 2.0 
                 2.4 
                 1.9 
                 2.2 
               
               
                 18C 
                 PB 
                 444 
                 479 
                 2.7 
                 5.3 
                 2.1 
                 4.2 
               
               
                 140C  
                 B 
                 440 
                 552 
                 0.19 
                 0.22 
                 0.1 
                 0.12 
               
               
                 142C  
                 B 
                 483 
                 515 
                 0.2 
                 0.2 
                 0.12 
                 0.11 
               
               
                   
               
               
                   a Determined by fitting the ratiometric signal of the intensities measured at λ1 and λ2 to equations 1-5. 
               
               
                   b B, Badan; PB, Pacific Blue. 
               
               
                   c Calcium titration in the presence of 50 mM NaHCO 3 . 
               
            
           
         
       
     
     Example 5. Sensor Engineering Phase 4: Affinity Tuning 
     The response of biosensors based on Langmuir binding isotherms is most sensitive at analyte concentrations that match the apparent K d  value of the protein (de Lorimier et al. 2002. Marvin et al. 1997). Accordingly sensor performance frequently needs to be optimized by “tuning” ligand affinity. The mutations that alter ligand affinities in PBPs generally fall into two classes:
         1. Alteration of direct interactions in the PCS between the protein and the bound bicarbonate.   2. Manipulation of the equilibrium between the open and closed states (Marvin and Hellinga 2001).   3. Indirect interactions that alter the geometry of the binding site.       

     Tuning of Bicarbonate Affinity in avBicarBP5. 
     Physiological blood bicarbonate levels for a healthy individual are typically between ˜20 mM to ˜30 mM (Burtis 2012). A series of mutations were introduced at positions 16 and 141, respectively, to obtain variants with K d  values near that concentration range (Table 10). Position 16 is located adjacent to the binding site, but does not interact directly with the bound complex. Mutations at this location therefore fall into the 3 rd  category. The threonine at position 141 (T192 in synBicarbBP1) forms a hydrogen bond with the bound bicarbonate ( FIG. 3C ). Mutations at this position therefore fall into the 1 st  category. The effects of these mutations were evaluated using ngmFRET in the doubly labeled C-terminal βZif fusions (Table 10). The I16F mutation in the 18C Pacific Blue background, combined with a Fluorescein acceptor, gives a response with a near-optimally tuned bicarbonate affinity ( FIG. 10 ). 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Bicarbonate response of βZif conjugates of the avBicarbBP5 scaffold a . 
               
            
           
           
               
               
               
               
            
               
                   
                 Emission 
                   
                   
               
               
                   
                 wavelength 
                 K d   b   
                 K d   c,e   
               
               
                 Conjugate 
                 (nm) 
                 (mM) 
                 (mM) 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Mutant 
                 avBicarbBP5 
                 βZif 
                 λ1 
                 λ2 
                   app K d   
                   true K d   
                   app K d   
                   true K d   
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 18C, 16M 
                 Pacific Blue 
                 5-IAF 
                 455 
                 520 
                 6 d   
                 6 d   
                 160 
                 170 
               
               
                 18C, 16M 
                 Badan 
                 Alexa532 
                 488 
                 555 
                   
                   
                 273 
                 243 
               
               
                 18C, 16F 
                 Pacific Blue 
                 5-IAF 
                 455 
                 517 
                 15 
                 20 
                 12 d   
                 13 d   
               
               
                 18C, 16F 
                 Badan 
                 Alexa532 
                 488 
                 555 
                   
                   
                 480 
                 700 
               
               
                 18C, 16F 
                 Pacific Blue 
                 Texas Red 
                 455 
                 620 
                 31 
                 25 
               
               
                 18C, 16Y 
                 Pacific Blue 
                 5-IAF 
                 455 
                 515 
                 10 
                 12 
                 34 
                 46 
               
               
                 18C, 16Y 
                 Badan 
                 Alexa532 
                 488 
                 555 
                   
                   
                 nb 
                 nb 
               
               
                 18C, 16W 
                 Pacific Blue 
                 5-IAF 
                 455 
                 520 
                 61 
                 64 
                 90 d   
                 70 d   
               
               
                 18C, 16W 
                 Badan 
                 Alexa532 
                 488 
                 555 
                   
                   
                 580 
                 780 
               
               
                 18C, 16E 
                 Pacific Blue 
                 5-IAF 
                 455 
                 520 
                 160 
                 130 
               
               
                 18C, 141F 
                 Pacific Blue 
                 5-IAF 
                 455 
                 520 
                 58 
                 69 
               
               
                 18C, 141F 
                 Badan 
                 Alexa532 
                 479 
                 555 
                   
                   
                 440 
                 430 
               
               
                 18C, 141Y 
                 Pacific Blue 
                 5-IAF 
                 455 
                 518 
                 5.4 
                 7.3 
                 nb 
                 nb 
               
               
                 18C, 141W 
                 Balan 
                 Alexa532 
                 483 
                 555 
                   
                   
                 nb 
                 nb 
               
               
                 18C, 141W 
                 Pacific Blue 
                 5-IAF 
                 455 
                 520 
                 35 
                 43 
                 2.6 
                 3.0 
               
               
                 18C, 141Q 
                 Pacific Blue 
                 5-IAF 
                 455 
                 520 
                 76 
                 96 
                 8.2 
                 9.3 
               
               
                 18C, 141Q 
                 Badan 
                 Alexa532 
                 488 
                 555 
                   
                   
                 770 
                 700 
               
               
                 18C, 141E 
                 Pacific Blue 
                 5-IAF 
                 455 
                 520 
                 196 
                 238 
                 138 
                 216 
               
               
                 190C 
                 Acrylodan 
                 5-IAF 
                 586 
                 460 
                 3.0 
                 3.4 
               
               
                 190C 
                 Acrylodan 
                 Alexa532 
                 487 
                 460 
                 5.5 
                 6.1 
               
               
                 194C 
                 Pacific Blue 
                 5-IAF 
                 455 
                 519 
                 24 
                 60 
               
               
                 194C 
                 Pacific Blue 
                 IANBD 
                 455 
                 519 
                 14 
                 61 
               
               
                 194C 
                 Pacific Blue 
                 Oregon 
                 455 
                 519 
                 24 
                 65 
               
               
                   
                   
                 Green 
               
               
                   
               
               
                   a Determined by fitting the ratiometric signal of the intensities measured at λ1 and λ2 to equation 1-5. 
               
               
                   b In the presence of 1 mM CaCl 2 . 
               
               
                   c In the presence of 1 mM EGTA. 
               
               
                   d Approximate value. 
               
               
                   e nb, no binding. 
               
            
           
         
       
     
     Tuning of Ca 2+  Affinity in avBicarbBP5. 
     The concentration of free Ca 2+  in blood is tightly regulated in the 1.2-1.3 mM range (Burtis 2012). The effect of mutations were studied at two labeled positions: 16C and 18C. Tuning mutations were introduced at positions 16 (in the 18C background), 49, 71, and 141, corresponding to positions 66, 99, 121, and 192, respectively in svnBicarbBP1. Each of affinity tuning positions therefore corresponds to position in the PCS ( FIG. 3C ). The effects of these mutations were evaluated using ngmFRET in the doubly labeled C-terminal βZif fusions (Table 11). The range of obtained Ca 2+  affinities spanned approximately five orders of magnitude, ranging from 20 μM to 360 mM, with each decade represented at least once. Several mutants exhibit K d  values appropriate for clinical sensing of ionized calcium ( FIG. 11 ). 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Calcium response of βZif conjugates of the avBicarbBP5 scaffold a . 
               
            
           
           
               
               
               
            
               
                   
                 Emission 
                   
               
               
                   
                 wavelength 
                 K d   
               
               
                 Conjugate 
                 (nm) 
                 (mM) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Mutant 
                 avBicarbBP5 
                 βZif 
                 λ1 
                 λ2 
                   app K d   
                   true K d   
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 16C 
                 Pacific Blue 
                 Oregon 
                 455 
                 520 
                 0.22 
                 0.26 
               
               
                   
                   
                 Green 
               
               
                 16C 
                 Pacific Blue 
                 5-IAF 
                 455 
                 517 
                 0.02 
                 0.02 
               
               
                 16C 
                 Alexa532 
                 5-IAF 
                 555 
                 515 
                 0.5 b   
                 0.5 b   
               
               
                 16C 
                 Alexa532 
                 Oregon 
                 551 
                 524 
                 13 b   
                 14 b   
               
               
                   
                   
                 Green 
               
               
                 16C, 71D 
                 Pacific Blue 
                 5-IAF 
                 455 
                 519 
                 19 b   
                 17 b   
               
               
                 16C, 71N 
                 Pacific Blue 
                 5-IAF 
                 455 
                 515 
                 0.21 
                 0.18 
               
               
                 16C, 71E 
                 Pacific Blue 
                 5-IAF 
                 455 
                 520 
                 380 
                 360 
               
               
                 16C, 71M 
                 Pacific Blue 
                 5-IAF 
                 455 
                 519 
                 20 
                 20 
               
               
                 17C 
                 Acrylodan 
                 5-IAF 
                 514 
                 465 
                 4.2 
                 1.8 
               
               
                 17C 
                 Badan 
                 5-IAF 
                 523 
                 465 
                 1.3 
                 0.51 
               
               
                 17C 
                 Acrylodan 
                 Alexa532 
                 483 
                 546 
                 2.6 
                 2.9 
               
               
                 17C 
                 Badan 
                 Alexa532 
                 491 
                 545 
                 0.85 
                 0.96 
               
               
                 17C 
                 Pacific Blue 
                 IANBD 
                 455 
                 483 
                 7.2 
                 18 
               
               
                 18C 
                 Pacific Blue 
                 5-IAF 
                 455 
                 523 
                 3.0 
                 3.1 
               
               
                 18C 
                 Pacific Blue 
                 Oregon 
                 455 
                 523 
                 3.3 
                 3.3 
               
               
                   
                   
                 Green 
               
               
                 18C 
                 Pacific Blue 
                 IANBD 
                 455 
                 480 
                 1.1 
                 1.7 
               
               
                 18C 
                 Acrylodan 
                 5-IAF 
                 523 
                 484 
                 8.9 
                 12 
               
               
                 18C 
                 Acrylodan 
                 Alexa532 
                 495 
                 470 
                 3.9 
                 4.1 
               
               
                 18C 
                 Badan 
                 Alexa532 
                 555 
                 500 
                 0.15 
                 0.20 
               
               
                 18C, 16M 
                 Pacific Blue 
                 5-IAF 
                 455 
                 517 
                 1.0 
                 0.91 
               
               
                 18C, 16M 
                 Badan 
                 Alexa532 
                 559 
                 491 
                 0.043 
                 0.076 
               
               
                 18C, 16F 
                 Pacific Blue 
                 5-IAF 
                 455 
                 520 
                 0.96 
                 1.1 
               
               
                 18C, 16F 
                 Badan 
                 Alexa532 
                 492 
                 555 
                 0.17 
                 0.18 
               
               
                 18C, 16F 
                 Pacific Blue 
                 Texas Red 
                 455 
                 620 
                 3.1 
                 3.0 
               
               
                 18C, 16Y 
                 Pacific Blue 
                 5-IAF 
                 455 
                 517 
                 0.77 
                 0.96 
               
               
                 18C, 16Y 
                 Badan 
                 Alexa532 
                 555 
                 490 
                 0.01 
                 0.02 
               
               
                 18C, 16Y 
                 Pacific Blue 
                 Texas Red 
                 455 
                 440 
                 16 
                 17 
               
               
                 18C, 16W 
                 Pacific Blue 
                 5-IAF 
                 455 
                 517 
                 0.76 
                 0.90 
               
               
                 18C, 16W 
                 Badan 
                 Alexa532 
                 487 
                 555 
                 0.02 b   
                 0.02 b   
               
               
                 18C, 16E 
                 Pacific Blue 
                 5-IAF 
                 455 
                 520 
                 0.20 
                 0.20 
               
               
                 18C, 49F 
                 Acrylodan 
                 Alexa532 
                 491 
                 547 
                 0.69 
                 0.84 
               
               
                 18C, 49F 
                 Pacific Blue 
                 Oregon 
                 455 
                 516 
                 19 
                 26 
               
               
                   
                   
                 Green 
               
               
                 18C, 49Y 
                 Pacific Blue 
                 Oregon 
                 455 
                 515 
                 15 
                 19 
               
               
                   
                   
                 Green 
               
               
                 18C, 49Y 
                 Badan 
                 Alexa532 
                 486 
                 555 
                 1.2 
                 1.4 
               
               
                 18C, 141V 
                 Acrylodan 
                 Alexa532 
                 487 
                 545 
                 9.7 
                 9.3 
               
               
                 18C, 141V 
                 Pacific Blue 
                 Oreon 
                 455 
                 518 
                 41 
                 40 
               
               
                   
                   
                 Green 
               
               
                 18C, 141F 
                 Pacific Blue 
                 5-IAF 
                 455 
                 522 
                 2.0 
                 2.1 
               
               
                 18C, 141F 
                 Badan 
                 Alexa532 
                 466 
                 555 
                 3.8 
                 4.0 
               
               
                 18C, 141Y 
                 Badan 
                 Alexa532 
                 467 
                 555 
                 2.0 
                 2.1 
               
               
                 18C, 141Y 
                 Pacific Blue 
                 5-IAF 
                 455 
                 520 
                 4.1 
                 4.5 
               
               
                 18C, 141W 
                 Badan 
                 Alexa532 
                 464 
                 555 
                 1.2 
                 1.3 
               
               
                 18C, 141W 
                 Pacific Blue 
                 5-IAF 
                 455 
                 522 
                 2.2 
                 2.0 
               
               
                 18C, 141Q 
                 Pacific Blue 
                 5-IAF 
                 455 
                 517 
                 1.7 
                 2.0 
               
               
                 18C, 141Q 
                 Badan 
                 Alexa532 
                 559 
                 490 
                 0.72 
                 0.57 
               
               
                 18C,141E 
                 Pacific Blue 
                 5-IAF 
                 455 
                 517 
                 1.4 
                 1.8 
               
               
                   
               
               
                   a Determined by fitting the ratiometric signal of the intensities measured at λ1 and λ2 to equation 1-5. 
               
               
                   b Approximate value. 
               
            
           
         
       
     
     Example 6. Materials and Methods 
     Bioinformatic Searches. 
     Annotated genomic and plasmid sequences of 5062 prokaryotes were obtained from the National Center of Biotechnology Information (ftp://ftp.ncbi.nih.gov/genomes/Bacteria/all.gbk.tar.gz), together with annotations recording prokaryotic lifestyles (../ProkaryotesOrganismInfo.txt). The Protein Databank (PDB) was obtained from www.rcsb.org. The downloaded genomic and structural data files were organized into pre-processed two databases (PG, prokaryotic genomes: PDB). The ‘ProteinHunter’ program provides an interface and methods for organizing, querying, and analyzing these databases. ProteinHunter comprises a graphical user interface, set of computer scripts, and a parallel computing environment. Together these set up the calculations, manage the flow of information and execution in each of the calculation phases, control other programs that carry out specific calculations such as BLAST (Altschul et al. 1990) and ClustalW (Chenna et al. 2003), and visualize the results. 
     To construct homolog sequence sets, single sequence seeds were extracted from either preprocessed PDB or PG databases. Homolog sets were then identified in the PDB or PG by using a seed sequence for a uni-directional BLAST search with the following parameters: expect threshold, 10.0; gap costs for existence, 11, and extension, 1: BLOSUM matrix; low complexity filter is on (the ProteinHunter package always executes BLAST searches with the following command “blastall -p blastp -m 8 -b 50000 -d&lt;database file&gt; -i&lt;input file&gt; -o &lt;output file&gt;, where &lt;database file&gt; specifies the name of the prebuilt search sequence file and &lt;input file&gt; and &lt;output file&gt; the seed sequence input and hit output files respectively. A pairwise BLAST alignment was scored in ProteinHunter as a homolog hit if it exceeded a minimum fraction of identical residues and if the alignment covered at least 70% of the probe and target sequences. 
     Function was inferred using the sequence of primary complementary surface (PCS) residues. A non-contiguous sequence comprising the PCS between the protein and the bound bicarbonate was identified using ProteinHunter, PCS residues were selected as members of the PCS if the calculated distance between any of their atoms and any bicarbonate atom was less than 5 Å, and the distances between their backbone C α  and any atom in bicarbonate-metal complex was greater than that of their C β  atom and any atom in the ligand complex. Secondary shell residues that do not form hydrogen bonds or van der Waals contacts were removed by inspection from the resulting set. To determine the PCS sequence of members in the homolog set identified in ProteinHunter, their sequences were aligned using ClustalW (Chenna et al. 2003). This alignment identifies the positions of the PCS residues in each homolog, from which the corresponding PCS sequence in that homology is then read. For each homolog, the number of PCS mutations relative to the bicarbonate-binding PCS (Hamming distance, H PCS ) was counted. Homologs with H PCS =0 were inferred to be bicarbonate-binding proteins. The PCS sequences were displayed sorted by their H PCS  values, and within each H PCS  value sorted by their fraction identical residues, indicating the replicon within which they reside (chromosome or plasmid), whether this replicon contains paralogs, and the temperature tolerance (hyperthermophile, thermophile, mesophile, psychrophile, unknown), their Gram stain classification (if known), and the percentage genomic AT content. Duplicate hits were removed automatically from this list if the organism name (genus and species), fractional identity and paralogs were the same. From this list representative, unique homologs with H PCS =0 were chosen by inspection. 
     Gene Synthesis and Mutagenesis. 
     The amino acid sequences for the predicted homologs identified in the bioinformatic search (see above) were extracted from the PG database. The putative leader peptide that mediates anchoring of the periplasmic-binding protein on the outside of the membrane (Gram positive bacteria) or directs secretion into the periplasm (Gram negative bacteria) was deleted by examining the multiple sequence alignment and removing the sequences N-terminal to the start of the mature seed protein amino acid sequence. Endogenous cysteines were changed to alanine. A hexahistidine tag was placed behind a GGS linker at the C-terminus of the mature protein to enable metal-mediated affinity purification (Hengen 1995). The final amino acid sequences were back-translated into a DNA sequence encoding the open reading frame (ORF), which was placed in a construct behind an efficient Shine-Dalgarno ribosome-binding site, and flanked by a T7 promoter and terminator at the 5′ and 3′ ends respectively, using the GeneFab program (Cox et al. 2007). The resulting ORF sequences were optimized in context by OrfOpt or OrfMorph programs designed to predict highly expressed mRNA sequences in  E. coli  (see below). The resulting DNA sequences were synthesized by oligonucleotide assembly and cloned into pUC57 by GeneWiz, Inc. (South Plainfield, N.J.). 
     Subsequent single and multiple point mutations were designed by preparing mutant sequences of the synthetic ORF sequences using the GfMutagenesis program that introduces point mutations into an ORF using the most prevalent codon in  E. coli  for an amino acid. Constructs for site-specific double labeling were designed by inserting the βZif domain sequence (Smith et al. 2005) before the hexa-histidine C-terminal purification tag. All variants also were constructed by total gene synthesis. 
     Synthetic Gene Optimization. 
     The OrfOpt program (U.S. Patent Publication No. 2011/0171737, incorporated by reference) uses stochastic optimization algorithms that choose different codons within an ORF without altering the amino acid sequence to optimize a target function designed to identify mRNA sequences that express proteins at high levels in  E. coli . The OrfOpt simultaneously imposes AU-rich nucleotide composition at the 5′ and 3′ ends of the ORF, low RNA secondary structure content and favorable codon usage (Allert et al. 2010). The OrfMorph program reproduces the pattern of codon usage and RNA secondary structure observed in the parent genome of a protein, but using  E. coli  codon preferences and nucleotide composition. 
     Codon usage is calculated using the codon adaptation index (CAI), as described for OrfOpt, using codon frequency tables calculated for the genome under examination. The mean CAI value for a genome, μ c , and its standard deviation, σ c , are calculated over all the codons in a genome. A codon usage score, c, is calculated for each codon in an open reading frame (ORF) by averaging the CAI over a 9-codon window, centered on the codon for which this score is calculated. A normalized codon usage score, z c , is calculated for each codon as Z-score: z c =(c−μ c )/σ c . A plot of z c  along an ORF establishes the codon usage pattern of that ORF. Rare codons (z c &lt;0) are hypothesized to slow down the elongation rate of ribosome translation, introducing “pause” sites at extreme values. Such pause sites are hypothesized to direct kinetics of co-translational folding, allowing a newly synthesized segment to fold before more protein is made. An RNA secondary structure score, s, is determined for each nucleotide by summing its participation in all possible hairpins that can form in its vicinity (settings: minimum duplex length 4 basepairs: maximum loop length, 30 bases: vicinity length, 100 bases), as described for OrfOpt. The average secondary structure energy, μ s , and its standard deviations, σ s . are calculated over all the nucleotides in a genome. A normalized secondary structure energy score, z s , is calculated for codon as the Z-score: z s =(c−μ s )/σ s . A plot of z s  along an ORF establishes the secondary structure pattern of that ORF. Regions of above-average secondary structure (z s &gt;0) are hypothesized to slow down the elongation rate of ribose translation, introducing “pause” sites at extremes. As with CAI-mediated pause sites, secondary structure-driven pause sites are hypothesized to direct the kinetics of co-translational folding. 
     To mimic these patterns for heterologous expression of an ORF in  E. coli , first the z, and z s  scores are calculated using the parent organism codon table, μ c , σ c , μ s , and σ s  values. Second, a stochastic search algorithm is used that randomly chooses between degenerate codons to construct trial mRNA nucleotide sequences, calculating z c  and z s  scores for each trial sequence, but using the  E. coli  codon table, and  E. coli μ   c , σ c , μ s , and σ s  values. For each trial, the absolute differences between the  E. coli  trial scores, and the wild-type scores are summed over the entire ORF. The OrfMorph program searches for a minimum of these differences. The stochastic search algorithm operates by first choosing a codon position, second choosing a degenerate codon within the allowed codons at that position. If the choice results in an improved score, the sequence is kept, otherwise it is rejected. After a position has been selected, it is removed from the pool of allowed positions, and the next is chosen from the remainder. The algorithm terminates when two successive sweeps do not yield further improvements in the score. The resulting RNA nucleotide sequence that has codon usage patterns and secondary structure patterns that closely match those of the wild-type mRNA sequence in its parental genomic context. The hypothesis is that such matching improves production of soluble protein by mimicking co-translational folding contributions that minimize mis-folded protein intermediate aggregation. 
     Protein Expression, Purification, and Fluorescent Conjugate Preparation. 
     Plasmids carrying the expression constructs (see above) were transformed into KRX competent cells (Promega), and grown overnight at 37° C. on LB agar plates (100 mg/mL ampicillin). For expression and purification of homologs of cyanobacterium  Synechocystis  PCC 6803 CaCl 2  (1 mM) was added. For expression and purification of homologs of  Mannheimia haemolytica  FeCl 3  (10 μM) was added. A single colony was picked and grown overnight at 37° C. in Terrific Broth (TB: Research Products International). The overnight cultures were diluted 1:20 in 500 mL TB (100 mg/mL ampicillin), grown to an optical density of A 600 =0.5 at 37° C. in vigorously aerated shaker flasks, induced by the addition of 2.5 mL rhamnose (20% w/v), and grown for a further 3-4 hrs. The cells were harvested by centrifugation (5,000 rpm, 10 min). After decanting the supernatant, the cell pellets were stored −80° C. The cell pellets were thawed, resuspended in 8 mL binding buffer (10 mM imadozole, 20 mM MOPS, 500 mM NaCl, pH 7.8). Following resuspension, 3 mL of BugBuster HT (EMD Millipore) was added. After incubation (20 mins, 25° C.), the cells were lysed on ice by sonication (2 minutes of one-second on/off pulses, 20-30% power). A clarified lysate was prepared by centrifugation (15,000 rpm, 20 min, 4° C.) from which recombinant protein was purified by batch immobilized metal affinity chromatography (IMAC). Resuspended IMAC agarose beads (5 mL: Sigma-Aldrich, P6611) were added to the lysate. After incubation at 4° C. in a Mini LabRoller (Labnet International) for 1 hr, the beads were washed at least five times with binding buffer. The immobilized protein beads were resuspended in labeling buffer (20 mM MOPS, 100 mM NaCl, pH 6.9) and labeled overnight (4° C., rotating end-over-end) with a thiol-reactive fluorophore (5-fold stoichiometric excess over protein). Following two rinses with labeling buffer to remove unincorporated label. For double labeling of βZif fusions, a second thiol-reactive label was added following reduction of the disulfide with 5 mM TCEP. To elute labeled protein from the IMAC beads, 6 mL of elution buffer (400 mM imidazole, 500 mM NaCl, 20 mM MOPS, pH 7.8) was added, incubated for 30 min (4° C., rotating end-over-end), and the beads removed by centrifugation. Following dialysis of the eluate against three changes of assay buffer (20 mM MOPS, 20 mM KCl, pH 7.4), using 10 kDa semi-permeable membrane (Snakeskin tubing, Thermo Scientific), the fluorescent conjugates were concentrated in a 10 kDa cutoff spin concentrator (Vivaspin, GE Healthcare). Protein purity was assessed by SDS/PAGE. Protein concentrations were determined by (Nanodrop1000) at 280 nm (using extinction coefficients calculated from their sequence (Gill and von Hippel 1989, Artimo et al. 2012)), or at the fluorophore absorbance peak (Acrylodan, 391 nm and Badan, 387 nm). 
     Preparation of Titration Series to Measure Ligand-Binding. 
     12-, 24-, or 48-point logarithmic titration series were prepared on a Tecan Freedom liquid-handling robot, using an in-house program, ‘TitrationPlate’, that compiles an abstract description of a multi-component titration series into machine instructions for operating the robot. For bicarbonate titrations in homologs of cyanobacterium  Synechocystis  PCC 6803, concentrations were varied from 0-1 M in 20 mM KCl, 20 mM MOPS (pH 7.4) supplemented with either 1 mM EGTA or 1 mM CaCl 2 . For bicarbonate titrations in homologs of  Mannheimia haemolytica , concentrations were varied from 0-1 M in 20 mM KCl, 20 mM MOPS (pH 7.4) in the presence of 10 μM FeCl 3 . For calcium titrations in homologs of cyanobacterium  Synechocystis  PCC 6803, concentrations were varied from 0-1 M in 20 mM KCl, 20 mM MOPS (pH 7.4), and for some experiments supplemented with 50 mM NaHCO 3 . 
     Determination of Temperature- and Ligand-Dependent Fluorescence Landscapes. 
     12-, 24-, or 48-point logarithmic titration series were prepared on a Tecan Freedom liquid-handling robot (see above). Temperature-dependent fluorescence emission intensities of 20 μL aliquots, each containing 10 μM protein, were measured in 384-well microtiter plates in a LightCycler 480 II (Roche) using excitation and emission wavelengths available for this instrument that most closely matched the optical characteristics of the fluorescent conjugate. Temperatures were advanced in 1K steps. At each temperature, data was collected at 1-second intervals for 60 seconds at which point the signal had relaxed to a steady value associated with the new temperature. Under these experimental photobleaching was not observed. The in-house program ‘TitrationMeltPlate’ was used to convert these observations into time-independent datasets that record fluorescence as a function of temperature for each well and associate wells with their concentration of titrant and additive. Management tools were developed to maintain a database of titrations and their analyses. 
     Determination of Emission Intensity Spectra. 
     Ligand- and wavelength-dependent emission intensities were recorded on a Nanodrop3300 (Thermo Scientific) at room temperature. Using the LED closest to the optimal excitation wavelength of the fluorophore (UV, 365 nm; blue, 470 nm; ‘white’, 460-550 nm). 
     Ratiometric Analysis of Ligand Binding. 
     Isothermal ligand titrations were extracted from the fluorescent landscape or emission spectra datasets obtained as described above. Monochromatic emission intensities I λ  (these intensities correspond to a bandpass intensity, recorded either with a physical filter in the case of the Roche LightCycler, or by integrating in the interval λ−δ, λ+δ in the case of an emission spectrum), were fit to 
         I   λ = apo β λ (1−   y     true )+ sat β λ     y     true   1
 
     where  apo β λ  and  sat β λ  are the fluorescence baselines associated with the ligand-free and ligand-bound states of the protein, respectively, and  y   true  the fractional saturation of the protein (Layton and Hellinga 2010). Baseline functions can be constant, linear, or a second-order polynomial. For the ligand- and temperature-dependent fluorescence landscapes, we use a constant value for  apo β r , but  sat β r  is described by a linear dependence on ligand concentration, [L]: 
     
       
         
           
             
               
                 
                   
                     β 
                     x 
                       
                       
                       
                     sat 
                   
                   = 
                   
                     
                       a 
                       x 
                     
                     + 
                     
                       
                         b 
                         x 
                       
                        
                       
                         [ 
                         L 
                         ] 
                       
                     
                   
                 
               
               
                 2 
               
             
           
         
       
     
     For a single ligand-binding site, the fractional saturation is given by 
     
       
         
           
             
               
                 
                   
                     y 
                     _ 
                   
                   = 
                   
                     
                       [ 
                       L 
                       ] 
                     
                     
                       
                         [ 
                         L 
                         ] 
                       
                       + 
                       
                         K 
                         d 
                       
                     
                   
                 
               
               
                 3 
               
             
           
         
       
     
     where [L] is the ligand (bicarbonate or calcium) concentration and K d  the dissociation constant,  true K d  for  y   true . 
     A ratiometric signal at a given point in a titration series, R 12 (t). is given by the ratio of intensities at two wavelengths,  obs I(λ 1 ,t),  obs I(λ 2 ,t) in the emission spectrum measured at that point: 
     
       
         
           
             
               
                 
                   
                     
                       R 
                       12 
                     
                      
                     
                       ( 
                       t 
                       ) 
                     
                   
                   = 
                   
                     
                       
                         a 
                         t 
                       
                        
                       
                         
                           
                               
                             obs 
                           
                            
                           I 
                         
                          
                         
                           ( 
                           
                             
                               λ 
                               1 
                             
                             , 
                             t 
                           
                           ) 
                         
                       
                     
                     
                       
                         a 
                         t 
                       
                        
                       
                         
                           
                               
                             obs 
                           
                            
                           I 
                         
                          
                         
                           ( 
                           
                             
                               λ 
                               2 
                             
                             , 
                             t 
                           
                           ) 
                         
                       
                     
                   
                 
               
               
                 4 
               
             
           
         
       
     
     where a t  is an attenuation factor that describes the effect of variations in sample size (i.e. the amount of observable fluorophore) in the i th  sample on the wavelength-independent intensity of the entire emission spectrum. This signal removes wavelength-independent emission intensity attenuation effects due to variations in conjugate concentration, photobleaching, fluctuations in excitation source intensities, and detection efficiency (Demchenko 2010, Demchenko 2014). It is a key aspect for high-precision sensing using the reagentless fluorescently-responsive sensors described here. The ratiometric signal also can be fit to a binding isotherm: 
         R   1,2 = apo β R (1−   y     R )+ sat β R     y     R   5
 
     where  apo β R  and  sat β R  are the baselines, and  y   R  the apparent fractional saturation of the protein (with  app K d ). In general,  true K d ≠ app K d : if both baselines are constant, a simple relationship can be derived relating  app K d  to  true K d  (Grimley et al. 2013): 
     
       
         
           
             
               
                 
                   
                     K 
                     d 
                       
                       
                       
                     app 
                   
                   = 
                   
                     
                       K 
                       d 
                         
                         
                         
                       true 
                     
                      
                     
                       
                         I 
                         
                           λ 
                            
                           
                               
                           
                            
                           2 
                         
                           
                           
                           
                         apo 
                       
                       
                         I 
                         
                           λ 
                            
                           
                               
                           
                            
                           2 
                         
                           
                           
                           
                         sat 
                       
                     
                   
                 
               
               
                 6 
               
             
           
         
       
     
     where  apo I λ2  and  sat I λ2  are the emission intensities of the monochromatic signal at wavelength λ 2  of the ligand-free and ligand-bound protein, respectively. 
     Following a fit of the titration series using equations 4 and 5, α t  values can be recovered by taking the average comparison of the observed and calculated intensities at the two wavelengths: 
     
       
         
           
             
               
                 
                   
                     a 
                     t 
                   
                   = 
                   
                     
                       1 
                       2 
                     
                      
                     
                       ( 
                       
                         
                           
                             
                               
                                   
                                 calc 
                               
                                
                               I 
                             
                              
                             
                               ( 
                               
                                 
                                   λ 
                                   1 
                                 
                                 , 
                                 t 
                               
                               ) 
                             
                           
                           
                             
                               
                                   
                                 obs 
                               
                                
                               I 
                             
                              
                             
                               ( 
                               
                                 
                                   λ 
                                   1 
                                 
                                 , 
                                 t 
                               
                               ) 
                             
                           
                         
                         + 
                         
                           
                             
                               
                                   
                                 calc 
                               
                                
                               I 
                             
                              
                             
                               ( 
                               
                                 
                                   λ 
                                   2 
                                 
                                 , 
                                 t 
                               
                               ) 
                             
                           
                           
                             
                               
                                   
                                 obs 
                               
                                
                               I 
                             
                              
                             
                               ( 
                               
                                 
                                   λ 
                                   2 
                                 
                                 , 
                                 t 
                               
                               ) 
                             
                           
                         
                       
                       ) 
                     
                   
                 
               
               
                 7 
               
             
           
         
       
     
     The a t  value can then be applied to all wavelengths to obtain an emission spectrum or integrated intensity of the t th  titration point corrected for variations in sample size: 
         corr   I (λ)= a   t   obs   I (λ)  8
 
     where  corr I(λ) and  obs I(λ) are the wavelength-dependent intensities of the corrected and observed emission spectra, respectively. 
     The fractional error in the chemometric concentration measurement, depends on the first derivative of the binding isotherm as follows (Marvin et al. 1997): 
     
       
         
           
             
               
                 
                   
                     
                       ∂ 
                       S 
                     
                     S 
                   
                   = 
                   
                     
                       
                         ɛ 
                         
                           1 
                           , 
                           2 
                         
                       
                       S 
                     
                     × 
                     
                       
                         ( 
                         
                           
                             dR 
                             
                               1 
                               , 
                               2 
                             
                           
                           
                             d 
                              
                             S 
                           
                         
                         ) 
                       
                       
                         - 
                         1 
                       
                     
                   
                 
               
               
                 9 
               
             
           
         
       
     
     Where R 1,2  is the ratiometric signal (equation 5), ε 1,2  its experimental error, and δS is the resulting chemometric error in the concentration. We can then define a relative precision function 
     
       
         
           
             
               
                 
                   
                     P 
                      
                     
                       ( 
                       S 
                       ) 
                     
                   
                   = 
                   
                     
                       S 
                       
                         δ 
                          
                         
                             
                         
                          
                         S 
                       
                     
                     × 
                     
                       1 
                       
                         P 
                         max 
                       
                     
                   
                 
               
               
                 10 
               
             
           
         
       
     
     where P(S) is the relative precision at concentration S, which reaches a maximum value (i.e. lowest error), P max , at the K d . 
     For a given isothermal titration, values for  app K d  and  true K d  were obtained using a non-linear fitting algorithm in which these two parameters were simultaneously fit to the three experimental binding isotherms using equations 1 and 5, with the two monochromatic isotherms sharing the same  true K d  value. Three separate pairs of  apo β and  sat β were fit in this procedure, corresponding to the two monochromatic and the ratiometric signals, respectively. Two distinct ratiometric response models can be used: coupled (both wavelengths respond to ligand): uncoupled (the second wavelength is non-responsive: i.e. remains constant). Optionally, an attenuation vector, a(t) containing a, values for each titration point (equation 7), can be refined by iterative fit cycles in which the a(t) vector of a previous cycle is used to adjust the integrated intensities of the next cycle. Programs ‘Nanodrop3300’ and ‘TitrationMeltAnalysis’ were developed to analyze wavelength- or temperature-dependent ligand-binding datasets respectively. 
     Analysis of Ligand-Binding Properties Using Thermal Melts. 
     The thermal stability of purified candidate proteins was determined by measuring the temperature-dependence of the fluorescence signal of an extrinsically added dye. SYPRO, using a Roche LightCycler (Layton and Hellinga 2010). The total fluorescence intensity, S, is given by 
         S=β   F   f   F +β U   f   U   11
 
     where f F  and f U  are the fractions of protein in the folded and unfolded states, respectively, and β F  and β U  the fluorescence baselines of these two states. To get the fractions of the two states, we have 
     
       
         
           
             
               
                 
                   
                     f 
                     N 
                   
                   = 
                   
                     
                       
                         1 
                         
                           1 
                           + 
                           
                             
                               K 
                               U 
                             
                              
                             
                               ( 
                               T 
                               ) 
                             
                           
                         
                       
                        
                       
                           
                       
                        
                       and 
                        
                       
                           
                       
                        
                       
                         f 
                         U 
                       
                     
                     = 
                     
                       1 
                       - 
                       
                         f 
                         N 
                       
                     
                   
                 
               
               
                 12 
               
             
           
         
       
     
     where K U (T) is the temperature-dependent unfolding equilibrium constant, which by the van′t Hoff approximation is given by 
     
       
         
           
             
               
                 
                   
                     K 
                     U 
                   
                   = 
                   
                     e 
                     
                       
                         - 
                         Δ 
                       
                        
                       
                           
                       
                        
                       
                         
                           
                             H 
                             U 
                           
                            
                           
                             ( 
                             
                               
                                 1 
                                 T 
                               
                               - 
                               
                                 1 
                                 
                                   T 
                                   m 
                                 
                               
                             
                             ) 
                           
                         
                         / 
                         R 
                       
                     
                   
                 
               
               
                 13 
               
             
           
         
       
     
     Where T is the temperature, T m , the unfolding reaction transition mid-point temperature, and ΔH U  the enthalpy of unfolding. 
     To obtain the temperature dependence of the binding reaction, the K d  values of all the individually determined isotherms were fit the Gibbs-Hemholtz equation (Layton and Hellinga 2010): 
     
       
         
           
             
               
                 
                   
                     Δ 
                      
                     
                         
                     
                      
                     
                       
                         G 
                         b 
                         • 
                       
                        
                       
                         ( 
                         T 
                         ) 
                       
                     
                   
                   = 
                   
                     
                       
                         Δ 
                         ref 
                       
                        
                       
                         H 
                         b 
                         • 
                       
                     
                     + 
                     
                       Δ 
                        
                       
                           
                       
                        
                       
                         
                           C 
                           
                             p 
                             , 
                             b 
                           
                         
                          
                         
                           ( 
                           
                             T 
                             - 
                             
                               T 
                               ref 
                             
                           
                           ) 
                         
                       
                     
                     - 
                     
                       T 
                        
                       
                         ( 
                         
                           
                             
                               Δ 
                               ref 
                             
                              
                             
                               S 
                               b 
                               • 
                             
                           
                           + 
                           
                             Δ 
                              
                             
                                 
                             
                              
                             
                               C 
                               
                                 p 
                                 , 
                                 b 
                               
                             
                              
                             
                                 
                             
                              
                             ln 
                              
                             
                                 
                             
                              
                             
                               T 
                               
                                 T 
                                 ref 
                               
                             
                           
                         
                         ) 
                       
                     
                   
                 
               
               
                 14 
               
             
           
         
       
     
     where ΔG b *(T) is the standard free energy of binding at 1 M ligand at temperature T, 
     
       
         
           
             
               
                 
                   
                     Δ 
                      
                     
                         
                     
                      
                     
                       
                         G 
                         b 
                         • 
                       
                        
                       
                         ( 
                         T 
                         ) 
                       
                     
                   
                   = 
                   
                     
                       - 
                       RT 
                     
                      
                     
                         
                     
                      
                     ln 
                      
                     
                         
                     
                      
                     
                       ( 
                       
                         1 
                         + 
                         
                           1 
                           
                             
                               K 
                               d 
                             
                              
                             
                               ( 
                               T 
                               ) 
                             
                           
                         
                       
                       ) 
                     
                   
                 
               
               
                 15 
               
             
           
         
       
     
     Δ ref H b * and Δ ref S b * the molar enthalpy and entropy of binding, respectively, at the reference temperature, T ref , and ΔC p,b  the heat capacity of the binding reaction. This data analysis was carried out using ‘TitrationMeltAnalysis’. 
     Mechanisms for Chemical Sensing Based on Non-Geometric Modulation of FRET. 
     The subject matter disclosed herein is not limited to or bound by any particular scientific theory. However, discussions regarding ngmFRET are provided to facilitate the understanding of possible mechanisms involved with ngmFRET signaling in various embodiments described herein. Equations for calculating various values mentioned herein are also provided. ngmFRET is also described in PCT International Patent Application No. PCT/US16/62958, filed Nov. 19, 2016, the entire content of which is incorporated herein by reference. 
     The total signal, S, of a fluorescent sensor (either single-wavelength emission intensities, I λ , or ratios of intensities at two wavelengths. R 12 ) is the sum of the fluorescence due to the ligand-free (apo) and ligand-bound states: 
         S =α(1− γ )+β   y     16
 
     where α and β are the fluorescent baselines in the ligand-free and -bound states, respectively, and  y  is the fractional occupancy of the binding sites (equation 3). 
     Fluorescence quantum yields are the fractions of photons emitted by the excited state relative to the total absorbed, and correspond to the ratio of the radiative decay rate relative to the sum of the rates of all possible decay pathways ( FIG. 12 ). For a single fluorophore: 
     
       
         
           
             
               
                 
                   Q 
                   = 
                   
                     
                       k 
                       r 
                     
                     
                       
                         k 
                         r 
                       
                       + 
                       
                         k 
                         nr 
                       
                     
                   
                 
               
               
                 17 
               
             
           
         
       
     
     where k r  and k nr  are the radiative and non-radiative decay rates of the excited state, respectively. If we define q as the ratio between the radiative and non-radiative decay rates. 
     
       
         
           
             
               
                 
                   q 
                   = 
                   
                     
                       k 
                       nr 
                     
                     
                       k 
                       r 
                     
                   
                 
               
               
                 18 
               
             
           
         
       
     
     then the quantum yield can be written as 
     
       
         
           
             
               
                 
                   Q 
                   = 
                   
                     1 
                     
                       q 
                       + 
                       1 
                     
                   
                 
               
               
                 19 
               
             
           
         
       
     
     Chemical sensors exploit the ligand-mediated shift of a fluorescent system between the ligand-free and ligand-bound states which each exhibit distinct quantum yields: 
         Q   obs   =Q   apo (1−   y   )+ Q   sat     y     20
 
     where Q obs , Q apo  and Q sat  are the quantum yield of the total system, the apo-protein, and the ligand-bound complex, respectively. In a system involving ngmFRET between a donor and acceptor fluorophore, the Q apo  and Q sat  quantum yields each are combinations of their respective donor and acceptor quantum yields: 
         Q   apo = D   Q   apo + A   Q   apo  and  Q   sat = D   Q   sat + A   Q   sat   21
 
     where the superscripts D and A indicate donor and acceptor fluorophores respectively. To understand ngmFRET-based sensors, we therefore need to examine the factors that affect each of these four quantum yields. 
     The intensity of the light emitted by a donor or its acceptor is determined by the rate of photon emission from their respective excited states ( FIG. 12A ). The excited state of a donor is formed by the incident light from the excitation source, and there are three pathways by which this state decays: radiative and non-radiative decay and resonance transfer (by itself and regardless of the presence of any other fluorophore/parter). By contrast, the rate of formation of the acceptor excited state is determined by the resonance transfer rate from the donor, and there are only two processes that determine its decay rate: the radiative and non-radiative pathways (by itself and regardless of the presence of any other fluorophore/parter). In an ngmFRET system, the patterns of ligand-mediated fluorescence intensity changes therefore depend on whether the fluorophore that responds directly to ligand binding functions as a donor or acceptor. To understand these relationships, we analyze the factors that determine the rates of formation and decay of the donor and acceptor excited states. 
     The rate of resonance energy transfer. k t , along a non-radiative pathway between donor and acceptor ( FIG. 12A ) is a fraction of the donor radiative emission pathway rate (by itself and regardless of the presence of any other fluorophore/parter),  D k r  (the emission rate in the absence of an acceptor) multiplied by the energy transfer coupling factor, ϕ, (Lakowicz 2006, Valeur 2012): 
         k   t   =φQ   D   D   n   k   22
 
     where Q D  is the donor quantum yield in the absence of an acceptor. 
     According to the Förster model of weakly coupled oscillators (Lakowicz 2006, Valeur 2012), the energy transfer coupling factor is dependent on the spectral overlap, J, of the donor emission,  D λ em , and acceptor excitation spectrum,  A λ ex , and the variation of the geometry, G, between the donor and acceptor excited state transition dipoles with distance, r, and orientation factor, κ. 
     
       
         
           
             
               
                 
                   ϕ 
                   = 
                   
                     
                       G 
                        
                       
                         ( 
                         
                           r 
                           , 
                           κ 
                         
                         ) 
                       
                     
                      
                     
                       J 
                        
                       
                         ( 
                         
                           
                             λ 
                             em 
                               
                               
                               
                             D 
                           
                           , 
                           
                             λ 
                             ex 
                               
                               
                               
                             A 
                           
                         
                         ) 
                       
                     
                      
                     
                       
                         9000 
                          
                         
                             
                         
                          
                         ln 
                          
                         
                             
                         
                          
                         10 
                       
                       
                         128 
                          
                         
                             
                         
                          
                         
                           π 
                           5 
                         
                          
                         
                           N 
                           A 
                         
                          
                         
                           n 
                           4 
                         
                       
                     
                   
                 
               
               
                 23 
               
             
             
               
                 where 
               
               
                 
                     
                 
               
             
             
               
                 
                   
                     G 
                      
                     
                       ( 
                       
                         r 
                         , 
                         κ 
                       
                       ) 
                     
                   
                   = 
                   
                     
                       κ 
                       2 
                     
                     
                       r 
                       6 
                     
                   
                 
               
               
                 24 
               
             
             
               
                 and 
               
               
                 
                     
                 
               
             
             
               
                 
                   
                     J 
                      
                     
                       ( 
                       
                         
                           λ 
                           em 
                             
                             
                             
                           D 
                         
                         , 
                         
                           λ 
                           ex 
                             
                             
                             
                           A 
                         
                       
                       ) 
                     
                   
                   = 
                   
                     ∫ 
                     
                       
                         F 
                          
                         
                           ( 
                           
                             λ 
                             em 
                               
                               
                               
                             D 
                           
                           ) 
                         
                       
                        
                       
                           
                       
                        
                       ɛ 
                        
                       
                           
                       
                        
                       
                         ( 
                         
                           λ 
                           ex 
                             
                             
                             
                           A 
                         
                         ) 
                       
                        
                       
                         λ 
                         4 
                       
                        
                       d 
                        
                       
                           
                       
                        
                       λ 
                     
                   
                 
               
               
                 25 
               
             
           
         
       
     
     with n the refractive index of medium, N A  Avogrado&#39;s number. F( D λ em ) the normalized donor emission spectrum, and ε( A λ ex ) the absorption coefficient of the acceptor excitation spectrum [this analysis is a re-arrangement of the traditional presentation of the equations describing traditional geometrically-modulated FRET (tgmFRET), separating the different contributions (geometry, spectral overlap, quenching)]. Ligand-mediated modulation of r, κ and J therefore affects k t  ( FIG. 12B-D ). leading to changes in donor and acceptor emission intensities (see below). 
     At steady state, the concentration of the donor excited state, [D*], is given by the following rate balance equation (see  FIG. 12A ): 
         N   0   αk   ex −[ D *]( D   k   nr + D   k   r   +k   t )=0  26
 
     where N 0  is the population of ground state fluorophores, k ex  the rate of excitation photon absorption, α the effective illumination, k t , the resonance energy transfer rate,  D k nr  and  D k r  the radiative and non-radiative decay rates of the donor (by itself and regardless of the presence of any other fluorophore/parter) in the absence of acceptor, respectively. Substituting  D k r (d+1) for  D k r + D k nr  (using equation 18, with d≡q, the ratio of non-radiative to radiative decay rates in the donor), and replacing k t  with equation 22 (with Q D =1/(1+d), according to equation 23), we obtain 
     
       
         
           
             
               
                 
                   
                     
                       
                         N 
                         0 
                       
                        
                       α 
                        
                       
                         k 
                         ex 
                       
                     
                     - 
                     
                       
                         
                           [ 
                           
                             D 
                             * 
                           
                           ] 
                         
                         D 
                       
                        
                       
                         
                           k 
                           r 
                         
                          
                         
                           ( 
                           
                             1 
                             + 
                             d 
                             + 
                             
                               ϕ 
                               
                                 1 
                                 + 
                                 d 
                               
                             
                           
                           ) 
                         
                       
                     
                   
                   = 
                   0 
                 
               
               
                 27 
               
             
             
               
                 Hense 
               
               
                 
                     
                 
               
             
             
               
                 
                   
                     [ 
                     
                       D 
                       * 
                     
                     ] 
                   
                   = 
                   
                     
                       
                         N 
                         0 
                       
                        
                       α 
                        
                       
                           
                       
                        
                       
                         k 
                         ex 
                       
                     
                     
                       
                         k 
                         r 
                           
                           
                           
                         D 
                       
                        
                       
                         ( 
                         
                           1 
                           + 
                           d 
                           + 
                           
                             ϕ 
                             
                               1 
                               + 
                               d 
                             
                           
                         
                         ) 
                       
                     
                   
                 
               
               
                 28 
               
             
           
         
       
     
     The intensity of the emitted donor light, I D , is 
     
       
         
           
             
               
                 
                   
                     I 
                     D 
                   
                   = 
                   
                     
                       
                         
                           [ 
                           
                             D 
                             * 
                           
                           ] 
                         
                         D 
                       
                        
                       
                         k 
                         r 
                       
                     
                     = 
                     
                       
                         
                           N 
                           0 
                         
                          
                         α 
                          
                         
                             
                         
                          
                         
                           k 
                           ex 
                         
                       
                       
                           
                         
                           ( 
                           
                             1 
                             + 
                             d 
                             + 
                             
                               ϕ 
                               
                                 1 
                                 + 
                                 d 
                               
                             
                           
                           ) 
                         
                       
                     
                   
                 
               
               
                 29 
               
             
           
         
       
     
     The donor quantum yield, Q D , is this emission intensity relative to the intensity of the excitation, k ex αN 0   
     
       
         
           
             
               
                 
                   
                     Q 
                     D 
                   
                   = 
                   
                     1 
                     
                         
                       
                         ( 
                         
                           1 
                           + 
                           d 
                           + 
                           
                             ϕ 
                             
                               1 
                               + 
                               d 
                             
                           
                         
                         ) 
                       
                     
                   
                 
               
               
                 30 
               
             
           
         
       
     
     The rate balance equation for the acceptor excited state concentration, [A*], is given by 
       └ D*┘k   t   −└A *┘( A   k   r + A   k   nr )  31
 
     Consequently, by applying equations 19, 22 and 30, the acceptor quantum yield, Q A , is 
     
       
         
           
             
               
                 
                   
                     Q 
                     A 
                   
                   = 
                   
                     ϕ 
                     
                       
                         ( 
                         
                           1 
                           + 
                           a 
                         
                         ) 
                       
                        
                       
                         ( 
                         
                           1 
                           + 
                           d 
                         
                         ) 
                       
                        
                       
                         ( 
                         
                           1 
                           + 
                           d 
                           + 
                           
                             ϕ 
                             
                               1 
                               + 
                               d 
                             
                           
                         
                         ) 
                       
                     
                   
                 
               
               
                 32 
               
             
           
         
       
     
     where a is the ratio of the radiative and non-radiative pathways in the acceptor. 
     The ratio of the acceptor and donor quantum yields therefore is 
     
       
         
           
             
               
                 
                   
                     
                       Q 
                       A 
                     
                     
                       Q 
                       B 
                     
                   
                   = 
                   
                     ϕ 
                     
                       
                         ( 
                         
                           1 
                           + 
                           d 
                         
                         ) 
                       
                        
                       
                         ( 
                         
                           1 
                           + 
                           a 
                         
                         ) 
                       
                     
                   
                 
               
               
                 33 
               
             
           
         
       
     
     This equation clearly shows that any ligand-mediated change in ngmFRET transfer (ϕ) or quenching of either the donor (d) or acceptor (a) leads to a change in the ratio of donor and acceptor emission intensities, thereby enabling ratiometry. 
     Classical ligand-mediated modulation of tgmFRET is concerned only with ligand-mediated changes in the distance between the donor and acceptor (Clegg 1995, Cheung 1991), and does not take advantage of effects that alter the photophysics of individual chromophores. By contrast, in ngmFRET systems, the directly responsive partner (DRP) responds to ligand binding through ligand mediated changes that alter the ratio of its radiative and non-radiative pathways (quenching, d or a) or its spectral properties (J), whereas the indirectly responsive partner (IRP) changes only as a consequence of the effect that such change have on the resonance energy transfer rate (k t ). It is important to realize that the DRP can function either as a ngmFRET donor an acceptor, depending on how the spectral overlap is set up with the IRP. Regardless of whether the DRP is a donor or acceptor, ligand-mediated alteration of its non-radiative to radiative decay rate ratio (parameter d for a DRP donor; a for an acceptor: by itself and regardless of the presence of any other fluorophore/parter) changes its emission intensity. In DRP donors quenching also alters the ngmFRET transfer rate (see equations 22 and 27), thereby changing the emission intensities of not only itself but also its IRP. By contrast, in DPR acceptors quenching does not alter ngmFRET, and hence do not affect its IRP donor intensity. A DRP acceptor therefore can alter intensities of its donor IRP only if ligand binding changes ϕ. If the DRP is a donor, then manipulation of the energy transfer coupling factor, ϕ, changes the rate of excited state decay; if it is an acceptor, the rate of excited state formation is altered. 
     Regardless of whether the DRP is a donor or acceptor, a change in any of the two parameters (ϕ and d or a) alters the ratio of the donor and acceptor quantum yields (equation 33), thereby enabling ratiometry. Ligand-mediated donor DRP quenching affects the quantum yields of both the donor, Q D , and acceptor, Q A , quantum yields (equations 30, 32). Quenching of an acceptor DRP alters only Q A  (equation 30). Changes in ϕ affect quantum yields of both fluorophores, regardless whether the DRP functions as the donor or acceptor (equations 23-25, 30, 32). For systems in which there is no ligand-mediated change in the (average) distance between the two fluorophores, ϕ changes only if the DRP switches between two different excited state populations (“dipole switching”) in response to ligand binding and if the two excited states differ in their spectral properties (emission for donor DRPs; absorption for acceptor DRPs). Excited state dipoles usually also differ in their dipole orientations, so it is likely that changes in spectral overlap involve (re-)orientation effects. They are also likely to differ in the relative rates of their radiative and non-radiative decay rates. Dipole switching therefore is likely to involve a combination of changes in ngmFRET and quenching effects. 
     There are eight possible combinations of ligand-mediated changes in quenching and ngmFRET parameters, which have different outcomes on the two emission intensities and their ratio, depending on whether the DRP is the donor or acceptor. The qualitative behavior of the resulting sixteen possibilities in ngmFRET systems are shown in Table 12. Twelve of these have a predictable outcome on the direction of change in the ratio of the two emission intensities. The effect on the direction of change for both donor and acceptor emission intensities can be predicted for seven models. For the other models, the direction of change of one or both peaks depends on the size of the change in the underlying parameters. Purely geometric effects (changes in inter-dipole distance or orientation) always result in anti-correlated changes in emission intensity changes (i.e. one increases and the other decreases, or vice versa). Correlated (i.e. both intensities increase or decrease) or uncorrelated (one changes, the other remains constant) intensity changes therefore are prima faice evidence for an ngmFRET effect. 
                     TABLE 12                  Qualitative analysis of the patterns of donor and acceptor        emission intensity changes in ngmFRET a                                   Directly                        responsive                        partner   Model   Q A /Q D     Q D     Q A                 Donor    d 0  ϕ +     ↑   ↓   ↑           d 0  ϕ −     ↓   ↑   ↓           d +  ϕ 0     ↓   ↓   ↓           d +  ϕ +     *   ↓   *           d +  ϕ −     ↓   *   ↓           d −  ϕ 0     ↑   ↑   ↑           d −  ϕ +     ↑   *   ↑           d −  ϕ −     *   ↑   *       Acceptor   α 0  ϕ +     ↑   ↓   *           α 0  ϕ −     ↓   ↑   *           α +  ϕ 0     ↓   0   ↓           α +  ϕ +     *   ↓   *           α +  ϕ −     ↓   ↑   *           α −  ϕ 0     ↑   0   ↑           α −  ϕ +     ↑   ↓   ↑           α −  ϕ −     *   ↑   *                      a The effects of increasing or decreasing quenching in the directly responsive ngmFRET partner (d for donors, a for acceptors) or the energy transfer coupling (ϕ) between the donor and acceptor are tabulated. The consequences of using a directly responsive donor or acceptor are examined. Changes in quenching and ngmFRET coupling parameters can occur singly or in combination, leading to 16 possible models. The models examine the effects of the direction of change in quenching parameters (no change, d 0  or a 0 ; increase d +  or a + ; decrease, d −  or a − ) and the energy transfer coupling factor (no change, ϕ 0 ; increase, ϕ + ; decrease, ϕ′) on the patterns in the direction of change of the donor. Q D  (equation 16) or acceptor, Q A  (equation 18) quantum yields, and their ratio, Q A /Q D  (equation 19): ↑, increase; ↓, decrease; 0, no change; *, response is dependent on precise quantitation rather than direction of change in the underlying parameter values.
 
     Example 7. Bicarbonate Biosensors, Calcium Biosensors, and Uses Thereof 
     We report the construction of robust, thermostable, reagentless, fluorescently responsive biosensors for bicarbonate and calcium, derived from engineered periplasmic binding proteins. These proteins potentially can be used for high-precision chemometric measurements in the clinical concentration ranges for bicarbonate and ionized calcium, using fluorescence ratiometry measured with straightforward, inexpensive instrumentation. 
     Thermostable homologs of the bicarbonate-binding protein were identified using a bioinformatics search strategy that applied a structure-based sequence filter to identify the subset of sequences that retain the original function within the larger collection of aligned sequence homologs. The homologs tested appeared at sequence identities from 100% to 26% of the probe sequence. At level below 60%, overall identities are weak predictors of biological function (Todd 2001, Tian 2003, George 2005), application of the structure-based filter therefore was essential for accurate identification. The bicarbonate-binding properties of the predicted hits were tested experimentally by constructing synthetic genes optimized for heterologous protein expression in  E. coli  (Allert et al. 2010) and determining the bicarbonate-binding properties of the expressed proteins. This search resulted in the identification of two thermostable proteins, avBicarbBP5 and teFeBP3, which bind Ca II - or Fe III -bicarbonate complexes, respectively. 
     In avBicarbBP5, Pacific Blue conjugates were particularly effective for the construction of bicarbonate sensors. Pacific Blue is a hydroxycoumarin derivative, the phenolate of which corresponds to the excited state (Sun 1998). Without being bound by any scientific theory, it is therefore likely that binding of Ca 2+  stabilizes the excited state, with a concomitant increase in quantum yield. We also found that Ca 2+  and bicarbonate binding functions can be separated. The avBicarbBP5 protein therefore can be engineered to function as either a Ca II —HCO 3  or a Ca 2+  sensor. The Pacific Blue conjugates exhibited monochromatic responses to ligand binding. These were converted into dichromatic responses that enable ratiometric measurements, using a double labeling strategy in which a second fluorophore was attached site-specifically to a small C-terminal βZif domain. Additional mutations further altered the affinities for either Ca II —HCO 3  or Ca 2+  to match the clinical concentration ranges of these two analytes. The resulting sensors are well-suited for applications in point-of-care clinical chemistry or continuous monitoring of analyte concentrations. 
     In teFeBP3, several singly labeled Acrylodan and Badan conjugates exhibited good dichromatic responses to Fe III —HCO 3  binding. Of these, the teFeBP3 270C-Badan conjugate evinced the largest response, with an apparent K d  value of 48 mM. This sensor is suitable for measurements in the clinical reference concentration range. 
     The biosensors can be incorporated into point-of-care clinical devices to measure ligand (e.g., bicarbonate or Ca 2+ ) concentrations accurately, and rapidly at the patient bedside. In such a device, a small blood sample (&lt;10 μL) is obtained by means of a finger stick using a lancet. This sample droplet is then placed on the aperture of a disposable cartridge containing desiccated, immobilized biosensors inside a small measurement chamber. The sample enters the chamber by virtue of passive capillary action, wetting the sensors upon contact. As soon as the sensors have been wetted, they bind ligand, and report on its concentration by virtue of the engineered fluorescent sensor mechanism. The cartridge is placed inside a small reader (handheld or on a desktop), and their fluorescence signal is measured by the (inexpensive) optoelectronic components of the reader. Excitation light is provided by a light-emitting diode (LED). In the case of Acrylodan or Badan, a commercially available 400 nm blue LED is used, and the emitted light is measured through two bandpass filters. Cartridges can contain multiple sensors, spanning the entire clinical range of possible ligand concentrations. Each sensor is immobilized at a particular, known location inside the cartridge, providing “spatial addressability”. The intensity at a particular wavelength is then recorded by imagining these sensors using an inexpensive camera, such as a Complementary metal-oxide semiconductor (CMOS) device commonly found in consumer electronics such as cell phones. Each pixel in the camera records the emitted light on a gray scale. Integration of that signal imaged through the two signals, is analyzed by an on-board computer to calculate the ratiometric signal for each immobilized sensor. Pre-recorded hyperbolic binding curves are then used to calculate the ligand concentration in the sample. Recording through multiple sensors, tuned for accurate detection at different ligand concentrations provides a high-accuracy reading. This process is completed in less than a minute. 
     Similar instrumentation can be used for any type of episodic measurements, for instance, using other bodily fluids, or samples obtained from animals, or non-biological samples such as foods and beverages. 
     The FRS biosensors also can be used to monitor ligand levels continuously. For instance, sensors can be immobilized at the tip of a thin optical fiber to construct a ligand-responsive optode. Such an optode can be introduced into the body subcutaneously, using a small needle. Excitation and emission light are passed to and from the immobilized sensor, respectively. The sensor is in continuous contact with the sample. Fluctuations in the ligand sample alter the dynamic equilibrium between the open and closed states of the ligand-binding protein, which is transduced into fluctuations of the fluorescent emission signal, by virtue of the sensing mechanism of the conjugated fluorophore. The emitted light intensities are read through filters by a reader connected to the optode. This reader continuously displays the change in signal, and the corresponding calculated ligand concentrations. Continuous ligand monitoring may be accomplished using a device containing the immobilized biosensor(s), e.g., a fiber optic biosensor, introduced into the subject intradermally or subcutaneously (Judge et al., 2011, Diabetes Technology &amp; Therapeutics 13 (3):309-317; Weidemaier et al., 2011, Biosensors and Bioelectronics 26:4117-4123; hereby incorporated by reference). 
     As was discussed above, the features that distinguish the described constructs, devices, and methods from earlier bicarbonate and Ca 2+  assay systems include:
         Self-calibration   Rapid response time   Simple sample-handling fluidic circuitry   No additional components/substrates (“reagentless”)   No incubation time to develop signal. Reading is near-instantaneous and continuous   Stability (simplifies manufacturing, distribution, storage)   Small sample volume (&lt;10 μL).   Capable of precise measurements over extended ligand concentration ranges (e.g., from low to high ranges)   Multiple sensors also provides redundancy, lowering error   Large scope of uses: episodic, continuous, ex vivo, in vivo, optodes, implants.       

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     Other Embodiments 
     While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 
     The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference. 
     While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.