# Patent

## Title
System and methods for the detection of multiple chemical compounds

## Abstract

Methods that may be used for the electrochemical detection of multiple parameters, including chemical compounds. Further provided are cells that may be used in the electrochemical detection of multiple parameters, including chemical compounds, as well as a kit for the electrochemical detection of multiple parameters, including chemical compounds.

## Background
RELATED APPLICATION
This application claims benefit of 35. U.S.C. 119 based on priority of U.S. Provisional Patent Application No. 61/880,277 filed on Sep. 20, 2013, which is incorporated herein in its entirety.


INCORPORATION OF SEQUENCE LISTING
A computer readable form of the Sequence Listing “21806-P44849US01_SequenceListing.txt” (36,864 bytes), submitted via EFS-WEB and amended on Oct. 23, 2014, is herein incorporated by reference.
FIELD OF THE DISCLOSURE
The present disclosure relates to methods for the detection of chemical compounds. More particularly, the present disclosure relates to methods for detecting chemical compounds using electrochemical means.
BACKGROUND OF THE DISCLOSURE
The following paragraphs are provided by way of background to the present disclosure. They are not however an admission that anything discussed therein is prior art or part of the knowledge of persons skilled in the art.
The reliable and rapid detection of chemical compounds in a liquid medium is desirable in many industrial processes, and detection methodologies uniquely relating a chemical property of a compound requiring detection to a detectable output signal are manifold. Recently, methods involving the use of reporter genes have been developed to detect the presence of chemical compounds. For example, the method disclosed in U.S. Pat. No. 6,391,549 permits the detection of a chemical compound using an electrochemical cell and a reporter polypeptide mediating the production of an electrically active compound. The method may be used to assay a single sample, or alternatively multiple samples may be assayed simultaneously. However the method is impractical where multiple chemical compounds require detection. In particular, in order to detect multiple chemical compounds, in accordance with the specification of U.S. Pat. No. 6,391,549, an electrical cell would be inserted in a first sample to detect a chemical compound, an assay result would be obtained, and then the electrical cell would be removed from the sample, cleaned and recalibrated for assaying for a second and any subsequent chemical compounds. Alternatively, two or more electrical cells are used in order to detect two or more chemical compounds. Thus, one of the drawbacks of the methods disclosed by U.S. Pat. No. 6,391,549, is that in order to detect the presence of multiple chemical compounds in an assay sample, it is required that either multiple assay samples are available, and, furthermore, that multiple electrochemical cells are employed, or that multiple non-simultaneous measurements are conducted. The former requires the presence of sufficient assay sample, and, moreover, requires a complex and impractical electrical component, in particular where a substantial number of different chemical compounds require detection. The latter is a time consuming methodology, and potentially more prone to operator error.
The reporter gene based methodologies for the detection of chemical compounds known to the prior art, including those provided by U.S. Pat. No. 6,391,549, are unsuitable for use in instances where it is desirable that multiple chemical compounds are detected in an assay sample. There exists therefore in the art a need for improved methods for the detection of chemical compounds.
SUMMARY OF THE DISCLOSURE
The following paragraphs are intended to introduce the reader to the more detailed description that follows and not to define or limited the claimed subject matter of the present disclosure.
The present disclosure relates to methods for the detection of multiple parameters in a liquid medium. Accordingly, the present disclosure provides, in at least one aspect, at least one embodiment of a method for simultaneously detecting two parameters in a liquid medium comprising:

    
    
        (i) providing a liquid medium comprising a first and second parameter;
        (ii) contacting in a vessel the liquid medium with cells and a first and second substrate to form an assay mixture, wherein the first substrate comprises a first electroactive analyte chemically linked to a first carrier and the second substrate comprises a second electroactive analyte chemically linked to a second carrier; and wherein the cells comprise a chimeric nucleic acid construct comprising in the 5′ to 3′ direction of translation as operably linked components a first nucleic acid construct comprising:
        
            (a) a first promoter inducible by the first parameter; and
            (b) a nucleic acid sequence encoding a first reporter polypeptide comprising a first hydrolase capable of hydrolyzing the first substrate and releasing the first electroactive analyte from the carrier; and
            a second nucleic acid construct comprising:
            (c) a second promoter inducible by the second parameter; and
            (d) a nucleic acid sequence encoding a second polypeptide comprising a second hydrolase capable of hydrolyzing the second substrate and releasing the second electroactive analyte from the carrier;
        
        
        (iii) detecting an electrical signal facilitated by the first and second electroactive analyte in the assay mixture, wherein detecting the electrical signal facilitated by the first and second electroactive analyte detects the first and second parameters in the liquid medium.
    
    


In another aspect, the present disclosure provides a cell comprising a chimeric nucleic acid sequence comprising in the 5′ to 3′ direction of translation as operably linked components a first nucleic acid construct comprising:

    
    
        (a) a first promoter operable in the cell; and
        (b) a nucleic acid sequence encoding a first reporter polypeptide comprising a first hydrolase capable of hydrolyzing a first substrate comprising a first electroactive analyte and a first carrier to release the first electroactive analyte from the carrier; and
        a second nucleic acid construct comprising:
        (c) a second promoter operable in the cell; and
        (d) a nucleic acid sequence encoding a second reporter polypeptide comprising a second hydrolase capable of hydrolyzing a second substrate comprising a second electroactive analyte and a second carrier to release the second electroactive analyte from the carrier.
    
    


In a preferred embodiment, the first promoter is an inducible promoter. In a further preferred embodiment, the first and the second promoter are inducible promoters.
In yet a further aspect, the present disclosure provides a mixture comprising two cells, the first cell comprising a first chimeric nucleic acid sequence comprising in the 5′ to 3′ direction of translation as operably linked components a first nucleic acid construct comprising:

    
    
        (a) a first promoter operable in the first cell; and
        (b) a nucleic acid sequence encoding a first reporter polypeptide comprising a first hydrolase capable of hydrolyzing a first substrate comprising a first electroactive analyte and a first carrier to release the first electroactive analyte from the carrier; and

the second cell comprising a second chimeric nucleic acid sequence comprising in the 5′ to 3′ direction of translation as operably linked components a first nucleic acid construct comprising:

        (c) a second promoter operable in the second cell; and
        (d) a nucleic acid sequence encoding a second reporter polypeptide comprising a second hydrolase capable of hydrolyzing a second substrate comprising a second electroactive analyte and a second carrier to release the second electroactive analyte from the carrier.
    
    


In yet a further aspect, the present disclosure relates to a method of detecting a first and second electroactive analyte in a liquid medium, the method comprising:

    
    
        (i) contacting in a vessel the liquid medium and (a) a first substrate comprising a first electroactive analyte chemically linked to a first carrier; (b) a second substrate comprising a second electroactive analyte chemically linked to a second carrier; (c) a first reporter polypeptide comprising a first hydrolase; and (d) a second reporter polypeptide comprising a second hydrolase, to form an assay mixture, wherein the first hydrolase is capable of hydrolyzing the first substrate and the second hydrolase is capable of hydrolyzing the second substrate to release the first and second substrate from the first and second carrier; and
        (ii) detecting an electrical signal facilitated by the first and second electroactive analyte in the assay mixture, wherein detecting the electrical signal facilitated by the first and second electroactive analyte detects the first and second parameters in the liquid medium.
    
    


In yet another aspect, the present disclosure also relates to a diagnostic kit and provides at least one embodiment of a diagnostic kit for simultaneously detecting two parameters comprising:

    
    
        (i) a cell comprising a chimeric nucleic acid sequence comprising in the 5′ to 3′ direction of translation as operably linked components a first nucleic acid construct comprising:
        
            (a) a first promoter operable in the cell; and
            (b) a nucleic acid sequence encoding a first reporter polypeptide comprising a first hydrolase capable of hydrolyzing a first substrate comprising a first electroactive analyte and a first carrier and releasing the first electroactive analyte from the carrier; and
        
        
         a second nucleic acid construct comprising:
        
            (c) a second promoter operable in the cell; and
            (d) a nucleic acid sequence encoding a second reporter polypeptide comprising a second hydrolase capable of hydrolyzing a second substrate comprising a second electroactive analyte and a second carrier and releasing the second electroactive analyte from the carrier;
        
        
        (ii) a first and second substrate; and
        (iii) instructions for use or storage of the kit.
    
    


Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description, while indicating preferred implementations of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those of skill in the art from the detailed description



BRIEF DESCRIPTION OF THE DRAWINGS
The disclosure is in the hereinafter provided paragraphs described in relation to its Figures. The Figures provided herein are provided for illustration purposes and are not intended to limit the present disclosure.
FIG. 1 depicts the chemical formulas of certain substrates and conversion thereof into electroactive analytes, notably the conversion of chlorophenol red-β-D galactopyranoside (CPRG) to chlorophenol red (CPR), para-nitrophenol-β-D-glucuronide (PNPG) to para-nitrophenol (PNP), para-diphenol-β-D-glucopyranoside (PDPG) to para-dihydroxyphenol (PDP).
FIG. 2 depicts a graph of a chronoamperometric measurement of the enzymatic conversion of the CPRG to CPR.
FIG. 3 depicts a graph of a chronoamperometric measurement of the enzymatic conversion of PDPG to PDP.
FIG. 4 depicts a graph of a chronoamperometric measurement of the enzymatic conversion of PNPG to PNP.
FIG. 5 depicts a voltammogram simultaneously measuring the in vivo enzymatic conversion of CPRG to CPR and PDPG to PDP.
FIG. 6 depicts a voltammogram simultaneously measuring the in vivo enzymatic conversion of PDPG to PDP, and PNPG to PNP.
FIG. 7 depicts a voltammogram, showing simultaneous detection of PDP, PNP and CPR. Arrows indicate the presence at unique voltages of each of the electroactive analytes.
FIG. 8 depicts a voltammogram measuring the in vivo enzymatic conversion of PDPG to PDP following induction of β-glucuronidase expression in E. coli. Various sweeps are shown. The ten minute (black), twenty minute (blue), and thirty minute (red) sweeps show the growth of a PDP peak in both maximum current and charge over time.
FIG. 9 depicts a voltammogram measuring the in vivo enzymatic conversion of PAPG to PAP following induction of β-galactosidase expression in E. coli. Various sweeps are shown. The baseline (black), ten minute (red), twenty minute (blue), and thirty minute (green) sweeps show the growth of a PAP peak in both maximum current and charge over time.
FIG. 10 depicts a voltammogram simultaneously measuring the in vivo enzymatic conversion of PAPG to PAP following induction of β-galactosidase expression and PNPG to PNP following induction of β-glucuronidase expression in E. coli. The baseline (black), ten minutes (blue), twenty minutes (green), and thirty minutes (red) sweeps show the growth of both PDP and PAP peaks in both maximum current and charge over time.
FIG. 11 depicts a differential pulse voltammogram simultaneously measuring the in vivo enzymatic conversion of PAPG to PAP following induction of β-galactosidase expression and PNPG to PNP following induction of β-glucuronidase expression in E. coli. The ten minute (black), twenty minute (blue), and thirty minute (red) minute sweeps are displayed to demonstrate the growth of both PDP and PAP reduction peaks in both maximum current and charge over time.
FIG. 12 depicts a graph showing the maximum current in the oxidation peak of PAP calculated and plotted as a function of time. Compared are E. coli cells induced to produce β-galactosidase and convert PAPG to PAP versus non-induced E. coli cells. Induced cells are represented by the blue diamonds and non-induced cells are represented by the red squares.
FIG. 13 depicts a voltammogram showing baseline production of PAP in E. coli comprising an inducible promoter under non-induced conditions. The ten (black), twenty (blue), and thirty (red) minute sweeps are displayed to demonstrate the minimal growth of a PAP peak in both maximum current and charge over time.



DETAILED DESCRIPTION OF THE DISCLOSURE
Various compositions or processes will be described below to provide an example of an embodiment of each claimed subject matter. No embodiment described below limits any claimed subject matter and any claimed subject matter may cover processes, compositions or systems that differ from those described below. The claimed subject matter is not limited to compositions or processes having all of the features of any one composition, system or process described below or to features common to multiple or all of the compositions, systems or processes described below. It is possible that a composition, system or process described below is not an embodiment of any claimed subject matter. Any subject matter disclosed in a composition, system or process described below that is not claimed in this document may be the subject matter of another protective instrument, for example, a continuing patent application, and the applicants, inventors or owners do not intend to abandon, disclaim or dedicate to the public any such subject matter by its disclosure in this document.
It should be noted that terms of degree such as “substantially”, “about” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of the modified term if this deviation would not negate the meaning of the term it modifies.
As used herein, the wording “and/or” is intended to represent an inclusive-or. That is, “X and/or Y” is intended to mean X or Y or both, for example. As a further example, “X, Y, and/or Z” is intended to mean X or Y or Z or any combination thereof.
All patents and patent applications, and other publications, cited herein, whether supra or infra, including nucleic acid and polypeptide sequences from GenBank, SwissPro and other databases, are hereby incorporated by reference in their entirety, where permitted.
It is further noted that, as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.
As hereinbefore mentioned, the present disclosure relates to methods for detecting multiple parameters in a liquid sample. The herein provided methods represent a novel and efficient means for detecting multiple parameters, including the presence of multiple chemical compounds. The methods of the present disclosure are particularly advantageous in that they permit the simultaneous detection of two or more compounds in a single vessel using a single electrical cell. To the best of the inventors' knowledge, the present disclosure provides, for the first time, a methodology involving the use of multiple reporter genes capable of facilitating the generation of separate electrical signals in response to the presence of two or more parameters in a single vessel using a single electrical cell. Thus a limited amount of assay sample is required in order to conduct the assay, and the electrical signal detection component of the assay is straightforward and inexpensive to manufacture and operate. Furthermore the source assay materials required for the assays are inexpensive and readily available, rendering the assays useful for implementation in a wide range of commercial applications.
Accordingly, the present disclosure provides, in at least one aspect, at least one embodiment of a method for simultaneously detecting two parameters in a liquid medium comprising:

    
    
        (i) providing a liquid medium comprising a first and second parameter;
        (ii) contacting in a vessel the liquid medium with cells and a first and second substrate to form an assay mixture, wherein the first substrate comprises a first electroactive analyte chemically linked to a first carrier and the second substrate comprises a second electroactive analyte chemically linked to a second carrier; and wherein the cells comprise a chimeric nucleic acid construct comprising in the 5′ to 3′ direction of translation as operably linked components a first nucleic acid construct comprising:
        
            (a) a first promoter inducible by the first parameter; and
            (b) a nucleic acid sequence encoding a first reporter polypeptide comprising a first hydrolase capable of hydrolyzing the first substrate and releasing the first electroactive analyte from the carrier; and
            a second nucleic acid construct comprising:
            (c) a second promoter inducible by the second parameter; and
            (d) a nucleic acid sequence encoding a second reporter polypeptide comprising a second hydrolase capable of hydrolyzing the second substrate and releasing second electroactive analyte from the carrier;
        
        
        (iii) detecting an electrical signal facilitated by the first and second electroactive analyte in the assay mixture, wherein detecting the electrical signal facilitated by the first and second electroactive analyte detects the first and second parameters in the liquid medium.
    
    


DEFINITIONS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.
The term “carrier” as used herein refers to a compound which can be chemically linked to an electroactive analyte and when linked thereto can be hydrolyzed, e.g. by a hydrolase, to form upon hydrolysis an electroactive analyte and the carrier.
The term “electroactive analyte” as used herein refers to a compound capable of being reduced and/or oxidized when a voltage is applied to an electrical cell and facilitate an electrical current in such electrochemical cell.
The term “hydrolase” as used herein refers to any and all enzymes comprising a sequence of amino acid residues which is (i) substantially identical to the amino acid sequences constituting any hydrolase polypeptide set fort herein, including, for example, SEQ.ID NO:2, SEQ. ID NO:4, SEQ.ID NO:6, or (ii) encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding any hydrolase polypeptide set forth herein, but for the use of synonymous codons.
The term “nucleic acid sequence” as used herein refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present disclosure may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil, and xanthine and hypoxanthine.
The herein interchangeably used terms “nucleic acid sequence encoding a hydrolase” and “nucleic acid sequence encoding a hydrolase polypeptide”, refer to any and all nucleic acid sequences encoding a hydrolase polypeptide, including, for example, SEQ.ID NO:1, SEQ.ID NO:3 and SEQ.ID NO:5. Nucleic acid sequences encoding a hydrolase polypeptide further include any and all nucleic acid sequences which (i) encode polypeptides that are substantially identical to the hydrolase polypeptide sequences set forth herein; or (ii) hybridize to any hydrolase nucleic acid sequences set forth herein under at least moderately stringent hybridization conditions or which would hybridize thereto under at least moderately stringent conditions but for the use of synonymous codons.
By the term “substantially identical” it is meant that two polypeptide sequences preferably are at least 70% identical, and more preferably are at least 85% identical and most preferably at least 95% identical, for example 96%, 97%, 98% or 99% identical. In order to determine the percentage of identity between two polypeptide sequences the amino acid sequences of such two sequences are aligned, using for example the alignment method of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443), as revised by Smith and Waterman (Adv. Appl. Math., 1981, 2: 482) so that the highest order match is obtained between the two sequences and the number of identical amino acids is determined between the two sequences. Methods to calculate the percentage identity between two amino acid sequences are generally art recognized and include, for example, those described by Carillo and Lipton (SIAM J. Applied Math., 1988, 48:1073) and those described in Computational Molecular Biology, Lesk, e.d. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects. Generally, computer programs will be employed for such calculations. Computer programs that may be used in this regard include, but are not limited to, GCG (Devereux et al., Nucleic Acids Res., 1984, 12: 387) BLASTP, BLASTN and FASTA (Altschul et al., J. Molec. Biol., 1990: 215:403). A particularly preferred method for determining the percentage identity between two polypeptides involves the Clustal W algorithm (Thompson, J D, Higgines, D G and Gibson T J, 1994, Nucleic Acid Res 22(22): 4673-4680 together with the BLOSUM 62 scoring matrix (Henikoff S & Henikoff, J G, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919 using a gap opening penalty of 10 and a gap extension penalty of 0.1, so that the highest order match obtained between two sequences wherein at least 50% of the total length of one of the two sequences is involved in the alignment.
By “at least moderately stringent hybridization conditions” it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm=81.5° C.−16.6 (Log 10 [Na+])+0.41(% (G+C)−600/1), or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1% mismatch may be assumed to result in about a 1° C. decrease in Tm, for example if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5° C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In preferred embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5× sodium chloride/sodium citrate (SSC)/5×Denhardt's solution/1.0% SDS at Tm (based on the above equation) −5° C., followed by a wash of 0.2×SSC/0.1% SDS at 60° C. Moderately stringent hybridization conditions include a washing step in 3×SSC at 42° C. It is understood however that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1.-6.3.6 and in: Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Vol. 3.
The term “chimeric” as used herein in the context of nucleic acid sequences refers to at least two linked nucleic acid sequences which are not naturally linked. Chimeric nucleic acid sequences include linked nucleic acid sequences of different natural origins. For example a nucleic acid sequence constituting a bacterial promoter linked to a nucleic acid sequence encoding a non-bacterial hydrolase protein is considered chimeric. Chimeric nucleic acid sequences also may comprise nucleic acid sequences of the same natural origin, provided they are not naturally linked. For example a nucleic acid sequence constituting a promoter obtained from a particular cell-type may be linked to a nucleic acid sequence encoding a polypeptide obtained from that same cell-type, but not normally linked to the nucleic acid sequence constituting the promoter, Chimeric nucleic acid sequences also include nucleic acid sequences comprising any naturally occurring nucleic acid sequence linked to any non-naturally occurring nucleic acid sequence.
The term “promoter” as used herein means any nucleic acid sequence element capable of conferring, effecting, initiating, directing, or enhancing transcription of a nucleic acid sequence element operably linked to the promoter, including, without limitation, SEQ.ID NO:7, SEQ.ID NO:8, SEQ.ID NO:9, SEQ.ID NO:10, SEQ.ID NO:11, SEQ.ID NO:12, SEQ.ID NO:13, SEQ.ID NO:14, SEQ.ID NO:15, SEQ.ID NO:16, SEQ.ID NO:17, SEQ.ID NO:18, and SEQ.ID NO:19. A promoter is generally located 5′ (i.e. upstream) of the transcribed nucleic acid sequence element. The transcribed nucleic acid sequence, in accordance herewith, corresponds with a coding region, and transcription thereof, in accordance herewith, effects the production of a polypeptide.
The terms “inducible promoter” or “promoter inducible by a parameter”, as may be used interchangeably herein, refer to a promoter which is activated by the presence of an exogenous stimulus, e.g. the presence of an exogenous parameter.
The term “constitutive promoter” as used herein refers to a promoter which provides transcription at a relatively constant rate independent from exogenous stimuli.
The term “reporter polypeptide” as used herein is a polypeptide that is used in order to mediate detection of a parameter.
The term “parameter” refers to the detectable existence of a condition in a liquid medium, including, without limitation, temperature, light, or the presence of a chemical compound.
General Implementation
In accordance with at least one embodiment of the present disclosure, one aspect of the methods herein provided involves contacting, preferably by mixing, in a liquid medium comprising two parameters, a first and second substrate, the first substrate comprising a first carrier and a first electroactive analyte, and the second substrate comprising a second carrier and a second electroactive analyte. The electroactive analyte in accordance herewith is a chemical compound which when free in a solution to which a voltage is applied, is capable of generating an electrical signal. Moreover when an electroactive analyte is chemically linked to the carrier and in solution, the application of a voltage to the solution does not generate a substantive electrical signal. In accordance herewith, the first and second electroactive analytes are non-identical chemical compounds. In further particularly preferred embodiments, the first and second electroactive analytes are selected such that they have distinct redox properties. By the term “distinct redox properties” it is meant that when a voltage is applied to the assay mixture in order to detect the electroactive analytes, as hereinafter described, such voltage application results in the generation of an electrical current through reduction and/or oxidation of the first and second electroactive analyte at voltages sufficiently separate from each other so that the amperage generated by the first electroactive analyte can be detected without substantive interference by the amperage generated by the second electroactive analyte. Thus the difference in voltage at which the first and second electroactive analyte provide an amperage is at least from about 0.1 Volt to about 0.5 Volt, more preferably at about 0.25 Volt to about 0.5 Volt, and in certain preferred embodiments, the difference in voltage at which the first and second electroactive analyte provide an amperage may be as low as 10 mV, e.g. between 0.01 V and 0.02 V. Electroactive analytes are further selected in such a manner that they do not damage or react chemically with the electrode that is used. Suitable electroactive analytes that may be used in accordance with the present disclosure are any electroactive analytes having a voltage range of approximately within +/−2 Volt when in an aqueous solution. Suitable electroactive analytes include independently selected phenolic compounds; single, double or triple ring aromatic compounds; phosphate conjugated compounds; chlorine or bromine substituted compounds; ring compounds comprising a double bond; indole-based compounds, cyanide based compounds; metal compounds; metal-conjugated organics (e.g. ferrocene); compounds with oxidizable alcohol groups; substituted naphthalene compounds; hydrazide based compounds, including 2-[4-(methylthio)phenyl]acetohydrazide; and triazole compounds and their derivatives, including -(4-methyltiobenzyl)-1,2,4-triazole-5-thione; and aromatic nitro compounds, such as para-nitrophenol (also known as 4-nitrophenol) (PNP). Further specific suitable electroactive analytes include, without limitation, para-diphenol (also known as 1,4-diphenol and as hydroquinone) (PDP), ortho-nitrophenol (ONP), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-GAL), chlorophenol-red (also known as 2-chloro-4-[3-(−chloro-4-hydroxyphenyl)-1,1-dioxobenzo[c]oxathiol-3-yl]phenol (CPR) bicarbazolyl), para-aminophenol (PAP), diazapam, ferrocyanide, nitrazepam, and papaverine. In preferred embodiments of the present disclosure, the electroactive analytes PNP, PDP, PAP and CPR are used.
The first and second carrier as used herein are chemical compounds which when chemically linked to an electroactive analyte are capable of being enzymatically hydrolyzed by a hydrolase. The first and second carrier selected may be chemically identical or may be chemically distinct. Suitable carriers that may be used in accordance herewith are any independently selected compounds that can function as a carrier, including any sugars and derivatives thereof. In preferred embodiments, the sugars used are glucose or a derivative thereof, including any oligosaccharide glucose linked to another molecule, phospho-glucose, any glucose-6-phosphate, glucose molecule modified with phosphate, nitrate, or any other chemical group, or galactose or a derivative thereof, including polygalactose, xylose, ribose, fructose, maltose, sucrose, chitin, any modified version of these chemicals, any disaccharide, any oligosaccharide, any orientation of these sugars D or L, any ketoses, aldose, any furanose or pyranose sugar type of any of the aforementioned sugars. Further carriers that may be used are glyceraldehyde, erythrose, threose, arabinose, lyxose, allose, altrose, mannose, gulose, idose, and talose. In particularly preferred embodiments, the carriers used in accordance herewith are β-glucopyranoside, β-galactopyranoside and β-D-glucoronide. Further carriers that may be used are mannoses and derivatives thereof; fructoses and derivatives thereof; fucoses and derivatives thereof; xyloses and derivatives thereof; rhamnoses and derivatives thereof; and chitin and derivatives thereof. Sugar linkages may be any beta- or alpha-linkage, including beta-1-4, beta-1-6, alpha-1-4 and alpha-1-6.
As hereinbefore mentioned, in accordance herewith the first and second carrier and first and second electroactive analyte are chemically linked to form a first and second substrate. Preferred substrates used in accordance herewith are chlorophenol red-β-D-galactopyranoside (CPRG), para-nitrophenol-β-D-glucuronide (PNPG), para-di-phenol-β-D-glucopyranoside (also known as arbutin) (PDPG) and para-aminophenol-β-galactopyranoside (PAPG). The substrate may be added in any form to the liquid medium, for example in the form of a pure or substantially pure compound. In another embodiment, the substrate is produced by the cells of the present disclosure, for example by including in the chimeric nucleic acid sequence, one or more sequences encoding polypeptides capable of mediating the production of the substrate in the cells. In a further embodiment in accordance herewith, a first cell produces the substrate, and a second cell produces the hydrolase.
In accordance herewith, the liquid medium is further mixed with a first reporter polypeptide comprising a first hydrolase and second reporter polypeptide comprising a second hydrolase, wherein the first hydrolase is capable of hydrolyzing the first substrate and the second hydrolase is capable of hydrolyzing the second substrate. It is preferable that the hydrolases are independently selected in such a manner that the first hydrolase and the second hydrolase are specific for hydrolysis of the first and second substrate, respectively. With term “specific” it is meant that hydrolyis by the first hydrolase of the second substrate is less than 5% (w/w), more preferably less than 1% (w/w) and most preferably 0.1% or less, and that the hydrolysis by the second hydrolase of the first substrate is less than 5% (w/w), more preferably less than 1% (w/w) and most preferably 0.1% or less. Preferred hydrolases are polypeptides capable of hydrolyzing the substrates CPRG, PNPG, PAPG and PDPG. Thus in preferred embodiments, the hydrolases are selected from a β-galactosidase, a β-D-glucuronidase and a β-D-glucosidase, and more preferably the hydrolases selected from the β-galactosidase having SEQ.ID. NO:2, the β-D-glucoronidase having SEQ.ID. NO:4 and the β-D-glucosidase, having SEQ.ID NO:6. As illustrated in FIG. 1, the hydrolases β-galactosidase having SEQ.ID. NO:2, β-D-glucuronidase having SEQ.ID. NO:4 and β-D-glucosidase, having SEQ.ID NO:6. are capable of hydrolyzing CPRG, PNPG and PDGP, respectively. The first and second reporter polypeptide, in addition to a first and second hydrolase polypeptide, may comprise additional peptide sequence, for example, a detection tag sequence, or the first and second reporter polypeptide may be restricted to and consist of the first and second hydrolase.
Further hydrolases that may be used in accordance herewith include: agarases including α-agarase, and β-agarase; amylases including α-amylase, β-amylase, and isoamylase; arabinosidases including β-L-arabinosidase, and arabinan endo-1,5-α-L-arabinosidase; facto-N-biosidase; carageenases including t-carrageenase, κ-carrageenase, and λ-carrageenase; cellulases; cellobiosidases including, cellulose 1,4-β-cellobiosidase (non-reducing end), and cellulose 1,4-β-cellobiosidase (reducing end); ceramidases, including glucosulceramidase, galactosylceramidase galactosylgalactoyslglucosylceramidase, glycosylceramidase, and endoglycosylceramidase; chitinases; chitosanase; dextrinases, including cyclomaltodextrinase, mycodextranase, and limit dextrinase; epimerases including UDP-N-acetylglucosamine 2-epimerase, and UDP-N,N′-diacetylbacillosamine 2-epimerase; fructofuranosidases, including β-fructofuranosidase; fucosidases, including β-D-fucosidase, α-L-fucosidase, 1,2-α-L-fucosidase, 1,3-α-L-fucosidase, and 1,6-α-L-fucosidase; fructosidases, including fructan β-fructosidase, fructan β-(2,1)-fructosidase, and fructan β-(2,6)-fructosidase; furanosidases, including non-reducing end α-L-arabinofuranosidase, and β-galactofuranosidase, non-reducing end β-L-arabinofuranosidase; galactosaminidase, including α-acetyl-galactosamindase, β-acetyl-galactoseaminidase, endo-α-N-acetylgalactosaminidase, and endogalactosaminidase; galactanases, including arabinogalactan endo-β-1,4-galactanase, and galactan endo-β-1,3-galactanase; galactosidases, including α-galactosidase, β-galactosidase, 6-phospho-β-galactosidase, capsular-polysaccharide endo-1,3-α-galactosidase, blood-group-substance endo-1,4-β-galactosidase, keratan-sulfate endo-1,4-β-galactosidase, galactan endo-1,6-β-galactosidase, galactan 1,3-β-galactosidase, blood group B linear chain α-1,3-galactosidase, and blood group B branched chain α-1,3-galactosidase; galacturonases, including polygalacturonase; galacturonidases, including galacturan 1,4-α-galacturonidase, and exo-poly-α-galacturonosidase; protein O-GlcNAcase; glucanases, including endo-1,3(4)-β-glucanase, xyloglucan-specific endo-β-1,4-glucanase, and xyloglucan-specific exo-β-1,4-glucanase; glucosaminidases, including hyaluronglucosaminidase, α-N-acetyl-glucosamindase, Mannosyl-glycoprotein endo-β-N-acetylglucosaminidase, and exo-1,4-β-D-glucosaminidase, β-aspartyl-N-acetylglucosaminidase; glucosidases, including 1,4-α-glucosidase, oligo-1,6-glucosidase, α-glucosidase, β-glucosidase, amylo-α-1,6-glucosidase, glucan-endo-1,3-β-glucosidase, GDP-glucosidase, sucrose-α-glucosidase, glucan 1,3-β-glucosidase, glucan endo-1,3-α-glucosidase, glucan 1,6-α-glucosidase, glucan endo-1,2-β-glucosidase, Glucan 1,4-β-glucosidase, glucan endo-1,6-β-glucosidase, glucan 1,3-α-glucosidase, 6-phospho-β-glucosidase, steryl-β-glucosidase, 3-α-M-strictosidine β-glucosidase, mannosyl-oligosaccharide glucosidase, protein-glucosylgalactosylhydroxylysine glucosidase, 2-deoxyglucosidase, branched-dextran exo-1,2-α-glucosidase, amygdalin β-glucosidase, prunasin β-glucosidase, vicianin β-glucosidase, oligoxyloglucan β-glycosidase, maltose-6′-phosphate glucosidase, raucaffricine β-glucosidase, coniferin β-glucosidase, thioglucosidase; 4-hydroxy-7-methoxy-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl glucoside β-D-glucosidase, β-D-glucopyranosyl abscisate β-glucosidase, β-apiosyl-β-glucosidase, and hesperidin 6-O-α-L-rhamnosyl-β-D-glucosidase; glucuronidases, including β-glucuronidase, α-glucuronidase, hyaluronoglucuronidase, glucuronosyl-disulfoglucosamine glucuronidase, glycyrrhizinate β-glucuronidase, xylan α-1,2-glucuronosidase, and baicalin-β-D-glucuronidase; glycosylases including DNA-3-methyladenine glycosylase I, thymine-DNA glycosylase, DNA-deoxyinosine glycosylase, DNA-3-methyladenine glycosylase II, rRNA N-glycosylase, DNA-formamidopyrimidine glycosylase, uracil-DNA glycosylase, and double-stranded uracil-DNA glycosylase; heparanase; hexosaminidases, including β-N-hexosaminidase; idanases, including fucoidanase; idurunodases, including L-iduronidase; inulases; lactases; levanase; licheninase; dictyostelium lysozyme A; maltosidases, including glucan 1,6-α-isomaltosidase; maltotriosidases including dextran 1,6-α-isomaltotriosidase, and glucan 1,4-α-maltotriohydrolase; maltotetraohydrolases, including glucan 1,4-α-maltotetraohydrolase; maltohexaosidases, including glucan 1,4-α-maltohexaosidase; maltohydrolases including glucan 1,4-α-maltohydrolase; mannosidases including mannan 1,4-mannobiosidase, mannan endo-1,6-α-mannosidase, mannosyl-oligosaccharide 1,2-α-mannosidase, and glycoprotein endo-α-1,2-mannosidase; mannosidases, including α-mannosidase, β-mannosidase, mannan 1,2-(1,3)-α-mannosidase, mannan endo-1,4-β-mannosidase, mannosyl-oligosaccharide 1,3-1,6-α-mannosidase, mannan exo-1,2-1,6-α-mannosidase, mannosylglycoprotein endo-β-mannosidase, and 1,6-α-D-mannosidase; muramidases, including peptidoglycan β-N-acetylmuramidase; nucleosidases, including N-methyl nucleosidase, purine nucleosidase, inosine nucleosidase, uridine nucleosidase, AMP nucleosidase, NAD(+) nucleosidase, NAD(P)(+) nucleosidase, adenosine nucleosidase, ribosylpyrirnidine nucleosidase, deoxyribodipyrimidine endonucleosidase, NMN nucleosidase, adenosylhomocysteine nucleosidase, pyrimidine-5′-nucleotide nucleosidase, inosinate nucleosidase, 1-methyladenosine nucleosidase, and methylthioadenosine nucleosidase; octulosonidases including 3-deoxy-2-octulosonidase, and 3-deoxyoctulosonase; β-primeverosidase; β-porphyranase; pullulanases, including isopullulanase and neopullulanase; quercitrinase; rhamnosidases, including α-L-rhamnosidase, and β-L-rhamnosidase; sialadases, including exo-α-sialadase, and endo-α-sialidase; difructose-anhydride synthase; trehalases, including α-α-trehalase, and α-α-phosphotrehalase; xylanases, including endo-1,4-β-xylanase, endo-1,3-β-xylanase, xylan-1,4,-xylosidase, Xylan 1,3-β-xylosidase, glucuronoarabinoxylan endo-1,4-β-xylanase, and oligosaccharide reducing-end xylanase; and still further include hydrolases selected from mannosylglycerate hydrolase 4-α-D-((1→4)-α-D-glucano)trehalose trehalohydrolase, rhamnogalacturonan hydrolase, futalosine hydrolase, poly(ADP-ribose) glycohydrolase, polymannuronate hydrolase, oligoxyloglucan reducing-end-specific cellobiohydrolase, unsaturated rhamnogalacturonyl hydrolase rhamnogalacturonan galacturonohydrolase, levanbiohydrolases, 2,6-β-fructan 6-levanbiohydrolase, α-neoagaro-oligosaccharide hydrolase, protein ADP-ribosylarginine hydrolase, ADP-ribosyl-dinitrogen reductase hydrolase, rhamnogalacturonan rhamnohydrolase, α-D-xyloside xylohydrolase, gellan tetrasaccharide unsaturated glucuronyl hydrolase, and unsaturated chondroitin disaccharide hydrolase.
Further hydrolases that may be used in accordance herewith include carboxylesterase; arylesterase; triacylglycerol lipase; phospholipase A2; lysophospholipase; acetylesterase; acetylcholinesterase; actylcholine acetylhydrolase; cholinesterase; tropinesterase; pectinesterase; sterol esterase; chlorophyllase; thioester hydrolase including acetyl-CoA hydrolase; succinyl-CoA hydrolase; glutathione thiolesterase; choloyl-CoA hydrolase; phosphoric-monoester hydrolase; alkaline phosphatase; acid phosphatase; phosphoserine phosphatase; phosphatidate phosphatase; 5′-nucleotidase, 3′-nucleotidase; phytase; phosphatases including glucose phosphatase, tre-halose phosphatase, histidinol-phosphatase, phosphoprotein phosphatase, sugar-phosphatase, inositol-phosphate phosphatase, 4-nitrophenylphosphatase, protein-tyrosine-phosphatase, sorbitol-6-phosphatase; and pyridoxal phosphatase; phosphodiesterase I; phospholipase D; 3′5′-cyclic-nucleotide phosphodiesterase; dGTPase; sulfatases, including arylsulfatase, steryl-sulfatase, glycosulfatase, choline-sulfatase, and iduronate-2-sulfatase; prenyl-diphosphatase; sclareol cyclase; ether hydrolase; adenosylhomocysteinase; cholesterol-5,6-oxide hydrolase; peptidases, including aminopeptidase, cystinyl peptidase, tripeptide aminopeptidase, prolyl aminopeptidase, glutamyl peptidase, bacterial leucyl aminopeptidase, clostridial aminopeptidase, cytosol alanyl aminopeptidase, tryptophanyl aminopeptidase, methionyl aminopeptidase, aspartyl aminopeptidase, dipeptidases, serine-type carboxypeptidases, and metallocarboxypeptidases; cathepsin; ubiquitinyl hydrolase; chymotrypsin; trypsin; thrombin; glutaminase; asparaginase; amidase; urease; arylformamidase; biotinidase; aminoacylase; nicotinamidase; glutaminase; pentanamidase; barbiturase; lactamase; creatininase; enamidase; arginase; deaminases, including adenine deaminase, adenosine deaminase, ADP deaminase, ATP deaminase, CMP deaminase, CTP and deaminase guanosine deaminase; nitrilase; arylacetonitrilase; thiocyanate hydrolase; riboflavinase; inorganic diphosphatase; apyrase; nucleotide diphosphatase; FAD diphosphatase; ADP-sugar diphosphatase; thiamine-triphosphatase; UDP-sugar diphosphatase; adenylylsulfatase; alkylhalidase; haloacetate dehalogenase; atrazine chlorohydrolase; phosphoamidase; cyclamate sulfohydrolase; phosphonoacetate hydrolase; and trithionate hydrolase,
Still further hydrolases that may be used in accordance herewith are any hydrolases within the class of enzymes belonging to Enzyme Commission numbers 1 and 3, (EC1; EC3), and preferably those belonging to EC3. In more preferred embodiments enzymes belonging to EC 3.1 are used, including those belonging to EC 3.1.1, EC 3.1.2, EC 3.1.3, EC 3.1.4, EC 3.1.5, EC 3.1.6, and EC 3.1.7; EC 3.2; EC 3.3, including EC 3.3.1, EC 3.4, EC 3.4.11, EC 3.4.13, EC 3.4.16, EC 3.4.17, EC 3.4.18; EC 3.5, including EC 3.5.2, EC 3.5.3, EC 3.5.4, EC 3.5.5, 3.5.99; EC 3.6, including EC 3.6.1, EC 3.6.2; EC 3.8, including EC 3.8.1; EC 3.9, including EC 3.9.1; EC 3.10, including EC 3.10.1; EC 3.11, including EC 3.11.1; and EC 3.12, including EC 3.12.1.
In preferred embodiments hydrolases are selected from the class of enzymes belonging to EC 3.2.1 and 3.2.2, and more preferably, EC 3.2.1.1, EC 3.2.1.2, EC 3.2.1.3, EC 3.2.1.4, EC 3.2.1.6, EC 3.2.1.7, EC 3.2.1.8, EC 3.2.1.10, EC 3.2.1.11, EC 3.2.1.14, EC 3.2.1.15, EC 3.2.1.18, EC 3.2.1.20, EC 3.2.1.21, EC 3.2.1.22, EC 3.2.1.23, EC 3.2.1.24, EC 3.2.1.25, EC 3.2.1.26, EC 3.2.1.28, EC 3.2.1.31, EC 3.2.1.32, EC 3.2.1.33, EC 3.2.1.35, EC 3.2.1.36, EC 3.2.1.37, EC 3.2.1.38, EC 3.2.1.39, EC 3.2.1.40, EC 3.2.1.41, EC 3.2.1.42, EC 3.2.1.2, EC 3.2.1.43, EC 3.2.1.44, EC 3.2.1.45, EC 3.2.1.46, EC 3.2.1.47, EC 3.2.1.48, EC 3.2.1.49, EC 3.2.1.50, EC 3.2.1.51, EC 3.2.1.52, EC 3.2.1.53, EC 3.2.1.54, EC 3.2.1.55, EC 3.2.1.56, EC 3.2.1.57, EC 3.2.1.58, EC 3.2.1.59, EC 3.2.1.60, EC 3.2.1.61, EC 3.2.1.62, EC 3.2.1.63, EC 3.2.1.64, EC 3.2.1.65, EC 3.2.1.66, EC 3.2.1.67, EC 3.2.1.68, EC 3.2.1.70, EC 3.2.1.71, EC 3.2.1.72, EC 3.2.1.73, EC 3.2.1.74, EC 3.2.1.75, EC 3.2.1.76, EC 3.2.1.77, EC 3.2.1.78, EC 3.2.1.80, EC 3.2.1.81, EC 3.2.1.82, EC 3.2.1.83, EC 3.2.1.84, EC 3.2.1.85, EC 3.2.1.86, EC 3.2.1.87, EC 3.2.1.88, EC 3.2.1.89, EC 3.2.1.90, EC 3.2.1.91, EC 3.2.1.92, EC 3.2.1.93, EC 3.2.1.94, EC 3.2.1.95, EC 3.2.1.96, EC 3.2.1.97, EC 3.2.1.98, EC 3.2.1.99, EC 3.2.1.100, EC 3.2.1.101, EC 3.2.1.102, EC 3.2.1.103, EC 3.2.1.104, EC 3.2.1.105, EC 3.2.1.106, EC 3.2.1.107, EC 3.2.1.108, EC 3.2.1.109, EC 3.2.1.110, EC 3.2.1.111, EC 3.2.1.112, EC 3.2.1.113, EC 3.2.1.114, EC 3.2.1.115, EC 3.2.1.116, EC 3.2.1.117, EC 3.2.1.118, EC 3.2.1.119, EC 3.2.1.120, EC 3.2.1.121, EC 3.2.1.122, EC 3.2.1.123, EC 3.2.1.124, EC 3.2.1.125, EC 3.2.1.126, EC 3.2.1.127, EC 3.2.1.128, EC 3.2.1.129, EC 3.2.1.130, EC 3.2.1.131, EC 3.2.1.132, EC 3.2.1.133, EC 3.2.1.134, EC 3.2.1.135, EC 3.2.1.136, EC 3.2.1.137, EC 3.2.1.138, EC 3.2.1.139, EC 3.2.1.140, EC 3.2.1.141, EC 3.2.1.142, EC 3.2.1.143, EC 3.2.1.144, EC 3.2.1.145, EC 3.2.1.146, EC 3.2.1.147, EC 3.2.1.149, EC 3.2.1.150, EC 3.2.1.151, EC 3.2.1.152, EC 3.2.1.153, EC 3.2.1.154, EC 3.2.1.155, EC 3.2.1.156, EC 3.2.1.157, EC 3.2.1.158, EC 3.2.1.159, EC 3.2.1.161, EC 3.2.1.162, EC 3.2.1.163, EC 3.2.1.164, EC 3.2.1.165, EC 3.2.1.166, EC 3.2.1.167, EC 3.2.1.168, EC 3.2.1.169, EC 3.2.1.170, EC 3.2.1.171, EC 3.2.1.172, EC 3.2.1.173, EC 3.2.1.174, EC 3.2.1.175, EC 3.2.1.176, EC 3.2.1.177, EC 3.2.1.178, EC 3.2.1.179, EC 3.2.1.180, EC 3.2.1.181, EC 3.2.1.182, EC 3.2.1.183, EC 3.2.1.184, EC 3.2.1.185, EC 3.2.1.n1, EC 3.2.1.n2, EC 3.2.1.n2, EC 3.2.1.2; and EC 3.2.2.1, EC 3.2.2.2, EC 3.2.2.3, EC 3.2.2.4, EC 3.2.2.5, EC 3.2.2.6, EC 3.2.2.7, EC 3.2.2.8, EC 3.2.2.9, EC 3.2.2.10, EC 3.2.2.11, EC 3.2.2.12, EC 3.2.2.13, EC 3.2.2.14, EC 3.2.2.15, EC 3.2.2.16, EC 3.2.2.17, EC 3.2.2.18, EC 3.2.2.19, EC 3.2.2.20, EC 3.2.2.21, EC 3.2.2.22, EC 3.2.2.23, EC 3.2.2.24, EC 3.2.2.25, EC 3.2.2.26, EC 3.2.2.27, EC 3.2.2.28, and EC 3.2.2.29.
In accordance herewith, cells comprising a chimeric construct comprising a promoter and a nucleic acid sequence encoding a reporter polypeptide comprising a hydrolase are prepared. Such cells may be any cells including, without limitation, any plant cells, animal cells, including human cells, or cells obtainable from or obtained from a microorganism, such as a bacterial cell or a fungal cell, e.g. Saccharomyces cerevisiae. Bacterial cells that may be used include Escherichia coli cells, Bacillus cells, Pseudomonas cells, Clostridium cells, Xanthamonas cells and Staphylococcus cells. Nucleic acid sequences may be introduced in the cell through a host cell vector suitable for expression in a host cell. The term “suitable for expression in a host cell” means that the host vector comprises genetic elements required to achieve expression of one or more polypeptides, including the reporter polypeptide comprising a hydrolase, encoded by the nucleic acid sequences introduced therein. Genetic elements that may be included in the host cell vector in this regard include a promoter, a transcriptional termination region, one or more nucleic acid sequences encoding marker genes, one or more origins of replication and the like. Generally, promoters obtained from bacterial cells are used when a bacterial host is selected in accordance herewith, while a fungal promoter is used when a fungal host is selected, a plant promoter will be used when a plant cell is selected, and so on.
In accordance herewith, a first and second promoter are selected, wherein the first and second promoter are inducible by the first and second parameter in the liquid medium. A wide variety of existing or novel promoters may be used in this regard. In preferred embodiments, the promoter is selected to be inducible by an environmental stimulus, including, without limitation: pH, the presence/absence of an organic compound, presence/absence of a toxic compound, pressure, light, sound, and the like. In a preferred embodiment, a nucleic acid sequence comprising the lacZ promoter is used, and more preferably the nucleic acid sequence comprising SEQ.ID. NO:7 is used. The lacZ promoter is induced by isopropyl-β-D-thiogalactopyranoside (IPTG) present in the liquid medium. Other promoters that may be used in accordance herewith include, any synthetically produced or naturally occurring promoter including: lac promoter (also known as Plac), Prha promoter (rhamnose inducible, glucose repressible), tet promoter (Ptet; tetracycline inducible), lambda cl promoter, Plas promoter, lux promoter (Ph.), lex promoter (Plex), PBad/araC promoter, omp promoter (Pomp), cinL/cinR promoter, PhaA (pH sensitive promoter) and a UV sensitive promoter; Further promoters include compound inducible promoters such as a copper sensitive promoter, including the cusR promoter (PcusR), and an iron sensitive promoter, including the fecA promoter (PfecA); further promoters that may be used include constitutive promoters a T7 promoter, σ24 promoter, σ28 promoter, σ32 promoter, σ38 promoter, and σ70 promoter (any sigma collection of promoter). Specific nucleic acid sequences of promoters that may be used in accordance herewith include SEQ.ID NO:8, SEQ.ID NO:9, SEQ.ID NO:10, SEQ.ID NO:11, SEQ.ID NO:12, SEQ.ID NO:13, SEQ.ID NO:14, SEQ.ID NO:15, SEQ.ID NO:16, SEQ.ID NO:17, SEQ.ID NO:18 SEQ.ID NO:19 SEQ.ID. NO: 20 and SEQ.ID NO: 21.
Additional novel inducible promoters may be identified, for example by screening a genetic library for such promoters. Thus, by way of example, in order to identify novel promoters that respond to a chemical compound (e.g. a toxic chemical compound) a library (e.g. a transposon library or a phage library) may be created in an organism (e.g. bacterial cells) known to metabolize the chemical compound. The transposon may be constructed to be a mobile genetic element containing a reporter gene (e.g. lacZ), a selectable marker (e.g. antibiotic resistance marker such as a tetracycline or ampicillin resistance marker), and the transposase enzyme responsible for gene insertion. By inserting the transposon randomly into the organism's genome a genetic library can be tested for inducible promoters. Isolated colonies, selected via the selectable marker cassette in the transposon, may be grown in the presence of the reporter gene and the chemical compound. Colonies in which the reporter gene is expressed comprise a promoter capable of induction by the chemical compound, which may be used to isolate the inducible promoter, or colonies may be used directly.
In further embodiments, the expression vector further may comprise genetic elements required for the integration of the vector or a portion thereof in the host cell's genome, for example if a plant host cell is used the T-DNA left and right border sequences which facilitate the integration into the plant's nuclear genome. Pursuant to the present disclosure the expression vector may further contain a marker gene. Marker genes that may be used in accordance with the present disclosure include all genes that allow the distinction of transformed cells from non-transformed cells, including all selectable and screenable marker genes. A marker gene may be a resistance marker such as an antibiotic resistance marker against, for example, kanamycin or ampicillin, or an auxotrophic marker. Screenable markers that may be employed to identify transformants through visual inspection include β-glucuronidase (GUS) and green fluorescent protein (GFP). One cell that particularly conveniently may be used is Escherichia coli. The preparation of the E. coli vectors may be accomplished using commonly known techniques such as restriction digestion, ligation, gel electrophoresis, DNA sequencing, the Polymerase Chain Reaction (PCR) and other methodologies well known to those of skill in the art. A wide variety of cloning vectors is available to perform the necessary steps required to prepare a recombinant expression vector. Among the vectors with a replication system functional in E. coli, are vectors such as pBR322, the pUC series of vectors, the M13 mp series of vectors, pBluescript etc. Typically, these cloning vectors contain a marker allowing selection of transformed cells. Nucleic acid sequences may be introduced in these vectors, and the vectors may be introduced in E. coli by preparing competent cells, electroporation, transfection or using other well known methodologies to a person of skill in the art. E. coli may be grown in an appropriate medium, such as Luria-Broth medium and harvested. Recombinant expression vectors may readily be recovered from cells upon harvesting and lysing of the cells. Further, general guidance with respect to the preparation of recombinant vectors and growth of recombinant cells, including, without limitation, E. coli, may be found in, for example: Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2012, Fourth Ed.
In another aspect, the present disclosure provides a cell comprising a chimeric nucleic acid sequence comprising in the 5′ to 3′ direction of translation as operably linked components a first nucleic acid construct comprising:

    
    
        (a) a first promoter operable in the cell; and
        (b) a nucleic acid sequence encoding a first reporter polypeptide comprising a first hydrolase capable of hydrolyzing a first substrate comprising a first electroactive analyte and a first carrier and releasing the first electroactive analyte from the carrier; and
    
    


a second nucleic acid construct comprising:

    
    
        (c) a second promoter operable in the cell; and
        (d) a nucleic acid sequence encoding a second reporter polypeptide comprising a second hydrolase capable of hydrolyzing a second substrate comprising a second electroactive analyte and a second carrier and releasing the second electroactive analyte from the carrier.
    
    


The first and second promoter may be a constitutive promoter or an inducible promoter. In a preferred embodiment, the first promoter is an inducible promoter. In a further preferred embodiment the first and the second promoter are inducible promoters. The cell may be an isolated or more or less pure cell or a cell cultured or suspended in a medium, solution, or growth medium.
In yet a further aspect, the present disclosure provides a mixture comprising two cells, the first cell comprising a first chimeric nucleic acid sequence comprising in the 5′ to 3′ direction of translation as operably linked components a first nucleic acid construct comprising:

    
    
        (a) a first promoter operable in the first cell; and
        (b) a nucleic acid sequence encoding a first reporter polypeptide comprising a first hydrolase capable of hydrolyzing a first substrate comprising a first carrier and a first electroactive analyte and releasing the first electroactive analyte from the carrier; and

the second cell comprising a second chimeric nucleic acid sequence comprising in the 5′ to 3′ direction of translation as operably linked components a first nucleic acid construct comprising:

        (c) a second promoter operable in the second cell; and
        (d) a nucleic acid sequence encoding a second reporter polypeptide comprising a second hydrolase capable of hydrolyzing a second substrate comprising a second carrier and a second electroactive analyte and releasing the second electroactive analyte from the carrier.
    
    


The first and second cells may be similar or even identical, but for the presence of the chimeric nucleic acid sequences therein, thus the first and second cells may each be E. coli cells, from the same or different strains. The cells also may be a mixture of two cell cultures comprising two different biological species. It will be clear to those of skill in the art that the mixture may comprise a plurality of cells genetically identical to the first and second cell.
Growth of the cells leads to production of the first and second hydrolase. In one embodiment, the hydrolase polypeptides may be recovered, isolated and separated from other components of the cell by a variety of different protein purification techniques including, e.g. ion-exchange chromatography, size exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, reverse phase chromatography, gel filtration, etc. Further general guidance with respect to protein purification may for example be found in: Cutler, P. Protein Purification Protocols, Humana Press, 2004, Second Ed. Thus a substantially pure protein may be obtained. By “substantially pure” it is meant that the protein is separated from other host cell components. In accordance here with the protein is at least 95% pure, and more preferably at least 96%, 97%, 98% or 99% pure. Such substantially pure hydrolase protein may be used to detect the presence of two or more electroactive analytes. Accordingly, the present disclosure in a further aspect includes an embodiment of a method of detecting a first and second electroactive analyte in a liquid medium, the method comprising:

    
    
        (i) contacting in a vessel the liquid medium and (a) a first substrate comprising a first electroactive analyte chemically linked to a first carrier; (b) a second substrate comprising a second electroactive analyte chemically linked to a second carrier; (c) a first reporter polypeptide comprising a first hydrolase; and (d) second reporter polypeptide comprising a second hydrolase, to form an assay mixture, wherein the first hydrolase is capable of hydrolyzing the first substrate and releasing the first electroactive analyte from the carrier and the second hydrolase is capable of hydrolyzing the second substrate and releasing the second electroactive analyte from the carrier; and
        (ii) detecting the electrical signal facilitated by the first and second electroactive analyte in the assay mixture, wherein detecting the electrical signal facilitated by the first and second electroactive analyte detects the first and second parameters in the sample.
    
    


In order to detect the first and second parameter in the sample, the electrical signal facilitated by the first and second electroactive analyte is detected and related to the presence of the first and second parameter. By the term “facilitated” it is meant that an electroactive analyte constitutes a conduit element of an electrical circuit, Thus the flow or conduct of an electrical current in a sample, to which a voltage is applied, is attained via an electroactive analyte, released from a carrier, resulting in the reduction or oxidation of the electroactive analyte.
The electrical signal may be detected in accordance herewith using any methodology involving the application of a voltage to the assay medium. The voltage may be applied potentiostatically (i.e. at one voltage) or in cyclic voltammetrical fashion (i.e. across a range of defined voltages). This further includes any voltamperometric methodology, including, without limitation, pulse voltammetry, linear sweep voltammetry, chromoamerometry, staircase voltammetry, and cyclical voltammetry, and variations or adaptations thereof such as differential pulse voltammetry, or wave based voltammetry with chronoamperometric steps included in the sweeps. In accordance herewith, the application of voltage to the assay medium results in the oxidation or reduction of the electroactive analyte and gain or release of electrons by the electroactive analyte or the electrode, which can be measured amperometrically in the form of a current. Voltages may be applied to a solution versus any stable reference electrode, including, without limitation, Ag/AgCl, saturated calomel electrode (SCE), saturated sodium chloride calomel electrode (SSCE), and the reduction of hydrogen electrode (RHE), and the voltage is between the production of oxygen from water at the positive end of the spectrum, and the production of hydrogen from water at the negative end. Voltages may be applied, for example, in the range from 0-2.0 V versus a reduction hydrogen electrode (RHE electrode) reference electrode, or −1 Volt to +1 Volt against a pseudo Ag/AgCl electrode. Amperages detected may range, for example, from 1 nA to 1 μA or more when a potentiostatic voltage is applied, or from about 1 nA to 10 μA or more when using cyclic voltammetry. Detection of the electrical signal in accordance with the herein disclosed methodologies involves the use of a single electrochemical cell, which is introduced in a single vessel, e.g. a single flask, tube, beaker, well plate or any other receptacle of any geometry or shape, comprising the assay mixture. The electrical cell generally comprises one or more working electrodes, a reference electrode, and a counter electrode. Further generally included is a potentiostat to control the electric cell. The composition of each of the electrodes in the electrochemical cell may vary, and depending on the composition of the reference electrode, the voltage measurements may change. The reference electrode may be any electrode that holds a consistent voltage when placed into the electrochemical cell, and is suitably a Ag/AgCl, saturated calomel electrode (SCE), or a saturated sodium chloride calomel electrode (SSCE). The counter electrode may, for example, be a gold or platinum electrode, or a carbon electrode, e.g. a printed carbon, glassy carbon or Vulcan carbon electrode. The composition of the working electrode may vary in composition, and, for example, be gold, platinum or carbon. Examples of preferred combinations of electrodes include: gold working electrode, reduction of hydrogen reference electrode, and a platinum counter electrode; glassy carbon working electrode, carbon counter electrode, and Ag/AgCl reference electrode; and platinum working electrode, gold counter electrode, SCE reference electrode. It is further advantageous that the electrode is coated e.g. by thiolate self-assembled monolayers on a metal surface (e.g. gold), and/or protected, for example, a carbon electrode may be protected by applying phthalocyanine layer, by application of certain ions or metals, e.g. nickel, which may be dried on the electrode surface or platinum which may be plated on the surface. Combinations of the foregoing may also be applied.
The electrochemical cell may be calibrated prior to initiating detection. This may conveniently be done using solutions comprising the first and second electroactive analyte. Voltage can be applied through various techniques, such as cyclic voltammetry, pulse voltammetry, square wave voltammetry, potentiostatically or any other form of voltamperometric methodologies and techniques. Amperages may be detected and obtained as desired, for example in the form of a voltammogram, or chronoamperometrically. Thus, it will be clear to those of skill in the art, that in accordance with the present disclosure, the first and second parameter in the assay medium are detected by relating the electrical signal to the presence of the first and second parameter in the assay mixture. As hereinbefore mentioned, voltages are preferably selected in accordance with the distinct redox properties of the electroactive analytes. In certain embodiments, an electrical signal is only detected once per assay mixture. In other embodiments hereof, a plurality of electrical signals is detected either at regular or irregular time intervals, or detection of the electrical signals may be conducted more or less continuously. For example, where the method of the present disclosure is used to detect a first and second nutritional compound in a growth medium in order to determine depletion thereof, repeated or continuous detection may be desirable. In yet another embodiment, samples are drawn from a liquid medium to form the assay mixture. It is further noted that detection using the methods of the present disclosure may be automated, and the electrochemical cell may be linked to a computer system to control the electrochemical cell and to record and analyze the electrical signal.
Flow of an electrical current and detection thereof, upon application of a voltage to the assay medium, thus signals the presence of the first and second parameter in the assay sample. Conversely, the absence of an electrical signal, upon the application of a voltage to the assay medium, is indicative of the absence of the first and second parameter in the assay sample. In this manner, the detection of the electrical signal, in accordance with the present disclosure, correlates with and detects the first and second parameter.
In yet another aspect, the present disclosure also relates to a diagnostic kit and provides at least one embodiment of a diagnostic kit for simultaneously detecting two parameters comprising:

    
    
        (i) a cell comprising a chimeric nucleic acid sequence comprising in the 5′ to 3′ direction of translation as operably linked components a first nucleic acid construct comprising:
        
            (a) a first promoter operable in the cell; and
            (b) a nucleic acid sequence encoding a first reporter polypeptide comprising a first hydrolase capable of hydrolyzing a first substrate comprising a first electroactive analyte and a first carrier and releasing the first electroactive analyte from the carrier; and
        
        
         a second nucleic acid construct comprising:
        
            (c) a second promoter operable in the cell; and
            (d) a nucleic acid sequence encoding a second reporter polypeptide comprising a second hydrolase capable of hydrolyzing a second substrate comprising a second electroactive analyte and a second carrier and releasing the second electroactive analyte from the carrier;
        
        
        (ii) a first and second substrate; and
        (iii) instructions for use or storage of the kit.
    
    


The kit may be provided in such a manner that it comprises one or more vessels, e.g. flasks, beakers, tubes, well plates, or microfluidic based device. The kit further may also be constructed in such a manner that it contains two cells: (I) a first cell comprising a chimeric nucleic acid sequence comprising in the 5′ to 3′ direction of translation as operably linked components a first nucleic acid construct comprising: a first promoter operable in the cell; and a nucleic acid sequence encoding a first reporter polypeptide comprising a first hydrolase capable of hydrolyzing a first substrate and producing a first electroactive analyte; and (II) a chimeric nucleic acid sequence comprising in the 5′ to 3′ direction of translation as operably linked components a second nucleic acid construct comprising a second promoter operable in the cell; and a nucleic acid sequence encoding a second reporter polypeptide comprising a second hydrolase capable of hydrolyzing a second substrate and producing a second electroactive analyte. The kit further may further be fabricated to comprise an electrochemical cell and/or a potentiostat.
Use of the Assays
The liquid medium as used herein can be any liquid medium including any aqueous solution, for example any buffer or salt solution, or any organic solution. In certain embodiments the medium may be an LB medium or more preferably an M9 minimal medium which are commonly used to grow E. coli bacterial cells. The first and second parameters in the liquid medium are two distinct parameters, for example, light and temperature, or ionic strength and temperature. In particularly preferred embodiments, the two parameters are two distinct chemical compounds. These compounds may be any chemical compounds and may be obtainable from any source, including from any liquid, gas or a solid, as desired. In embodiments where the compounds of interest are present in a liquid assay sample the compounds may be detected directly in the liquid sample, however where the compounds of interest are present in a gaseous sample or solid sample, the compounds are dissolved in a liquid medium prior to initiating detection thereof. As hereinbefore mentioned, the methods of the present disclosure permit the simultaneous detection of the presence of two parameters. It is noted however that the methodologies further permit the detection of a plurality of parameters, e.g. 3, 4, 5, 6, 7, 8, 9, 10 or more parameters. Thus when reference is made herein to a method involving the detection of a first and second parameter, it is intended that the methodology disclosed herein involves the detection of at least two parameters.
In further preferred embodiments, a first and second toxic compound found in the environment are detected, for example toxic compounds that may be present in e.g. in surface water or sediments, including for example compounds found in tailings ponds associated with mining or fossil fuel production, or toxic compounds associated with petroleum extraction, such as hydrocarbons and naphthenic acid, which may be released in the environment, for example, as a result of a pipeline or fracking well leak. In other embodiments, the chemical compounds are nutritional compounds used, e.g. nutritional compounds used by a microorganism culture, or toxins which accumulate in a microorganism's production system. It will be clear to those of skill in the art that the methodologies herein provided are independent of the exact parameters, including chemical compounds, to be detected. The first and second parameter, or the chemical formula of the first and second chemical compound does not matter and any chemical compound, or any parameter for which the presence of a compound serves as a proxy, may be detected in accordance with the methods provided by the instant disclosure.
EXAMPLES
Hereinafter are provided examples of specific embodiments for performing the methods of the present disclosure, as well as embodiments representing the compositions of the present disclosure. The examples are provided for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way.
Example 1—Detection of CPR Using Constitutively Expressed β-Galactosidase
For the potentiostatic detection of CPR as a product of the cleavage of CPRG by β-galactosidase, a solution of 0.1M pH 7 sodium phosphate buffer was used. 20 mL of this solution was placed into an electrochemical cell with a screen printed carbon electrode (Pine Instruments, USA) as the working electrode, a platinum mesh as the counter electrode, and a reduction of hydrogen reference electrode (RHE) immersed in an identical solution with constant hydrogen gas bubbling and connected via a luggin capillary. The buffer was bubbled with nitrogen for 15 minutes prior to use. 1 mL of an overnight culture of Pseudomonas fluorescens PFS constitutively expressing lacZ via a transposon insertion was pelleted and resuspended in 0.1M pH 7 phosphate buffer and then added to the electrochemical cell. The working electrode was then held at 1.325V vs RHE and the current allowed to stabilize for 1200 seconds. After stabilization the substrate CPRG was added to the solution (time zero in FIG. 2) and the current produced at the working electrode was recorded. FIG. 2 provides the results obtained.
Example 2—Detection of PNP Using Constitutively Expressed β-Glucuronidase
For the potentiostatic detection of PNP as a product of the cleavage of PNPG by β-glucuronidase, a solution of 0.1M pH 7 sodium phosphate buffer was used. 20 mL of this solution was placed into an electrochemical cell with a screen printed carbon electrode (Pine Instruments, USA) as the working electrode, a platinum mesh as the counter electrode, and a reduction of hydrogen reference electrode (RHE) immersed in an identical solution with constant hydrogen gas bubbling and connected via a luggin capillary. The buffer was bubbled with nitrogen for 15 minutes prior to use. 1 mL of an overnight culture of TOP10 E. coli expressing uidA under the control of the Lacl repressible promoter on a high copy number plasmid (PSB1C3) was pelleted and resuspended in 0.1M pH 7 phosphate buffer and then added to the electrochemical cell. One sample was treated with 500 μM IPTG during the overnight culture, while the second was grown without induction to assess basal (leaky) expression in the genetic circuit. The working electrode was then held at 1.6 V vs RHE for each sample and the current allowed to stabilize for 1200 seconds. After stabilization the substrate PNPG was added to the solution (time zero in FIG. 4) and the current produced at the working electrode was recorded. FIG. 4 provides the results obtained.
Example 3—Detection of PDP Using Inducibly Expressed β-Glucosidase
For the potentiostatic detection of PDP as a product of the cleavage of PDPG by β-glucosidase, a solution of 0.1M pH 7 sodium phosphate buffer was used. 20 mL of this solution was placed into an electrochemical cell with a screen printed carbon electrode (Pine Instruments, USA) as the working electrode, a platinum mesh as the counter electrode, and a reduction of hydrogen reference electrode (RHE) immersed in an identical solution with constant hydrogen gas bubbling and connected via a luggin capillary. The buffer was bubbled with nitrogen for 15 minutes prior to use. 1 mL of an overnight culture of TOP10 E. coli expressing native levels of bglX when grown in rich media was pelleted and resuspended in 0.1M pH 7 phosphate buffer and then added to the electrochemical cell. The working electrode was then held at 0.825V vs RHE and the current allowed to stabilize for 1200 seconds. After stabilization the substrate PDPG was added to the solution (time zero in FIG. 3) and the current produced at the working electrode was recorded. FIG. 3 shows the results obtained.
Example 4—Simultaneous Detection of PDP and CPR
For the simultaneous voltammetric detection of PDP and CPR at once as a product of the cleavage of PDPG and CPRG by β-glucosidase and β-galactosidase respectively, a solution of 0.1M pH 7 sodium phosphate buffer was used. 15 mL of this solution was placed into an electrochemical cell with a screen printed carbon electrode (Pine Instruments, USA) as the working electrode, counter electrode, and a screen printed Ag/AgCl pseudo-reference electrode immersed in the same solution. The buffer was bubbled with nitrogen for 15 minutes prior to use. 2 mL of an overnight culture of BL-21 E. coli was pelleted and resuspended in 0.1M pH 7 phosphate buffer and then added to the electrochemical cell. PDPG and CPRG were added to the solution and the working electrode was then swept between −1 V and 1 V vs the Ag/AgCl reference at 100 mV/s for 100 cycles. Six cycles 800 seconds apart, from the initial start of the sweep considering it to be the baseline (0 seconds), are reported in FIG. 5. Oxidation of PDP and CPR are noted (−0.2 and 0.4V respectively) as shown by the increase of peak heights after each voltammetric sweep.
Example 5—Simultaneous Detection of PDP and PNP
For the simultaneous in vivo voltammetric detection of PDP and PNP at once as a product of the cleavage of PDPG and PNPG by β-glucosidase and β-glucuronidase respectively, a solution of 0.1M pH 7 sodium phosphate buffer was used. 15 mL of this solution was placed into an electrochemical cell with a screen printed carbon electrode (Pine Instruments, USA) as the working electrode, counter electrode, and a screen printed Ag/AgCl pseudo-reference electrode immersed in the same solution. The buffer was bubbled with nitrogen for 15 minutes prior to use. 2 mL of an overnight culture of BL-21 E. coli with a plasmid harbouring the uidA gene under the control of an uninduced LacI responsive promoter was pelleted and resuspended in 0.1M pH 7 phosphate buffer and then added to the electrochemical cell. PDPG and PNPG were added to the solution and the working electrode was then swept between −1 V and 1 V vs the Ag/AgCl reference at 100 mV/s for 100 cycles. Six cycles 800 seconds apart, from the initial start of the sweep considering it to be the baseline (0 seconds), are reported in FIG. 6. Oxidation of PDP and PNP are noted (−0.2 and 0.7V respectively) as shown by the increase of peak heights after each sweep.
Example 6—Simultaneous Detection of PDP, CPR and PNP
For the simultaneous in vivo voltammetric detection of PDP, CPR, and PNP at once, a solution of 0.1M pH 7 sodium phosphate buffer was used. 15 mL of this solution was placed into an electrochemical cell with a screen printed carbon electrode (Pine Instruments, USA) as the working electrode, counter electrode, and a screen printed Ag/AgCl pseudo-reference electrode immersed in the same solution. The buffer was bubbled with nitrogen for 15 minutes prior to use. 0.2 mL of 2 mM PDP and PNP was added to the solution as well as 0.5 mL of 0.001% (w/v) CPR. After mixing the working electrode was then swept between −1 V and 1 V vs the Ag/AgCl reference at 100 mV/s for 3 cycles. Results are shown in FIG. 7.
Example 7—Detection of PDP Using Inducibly Expressed β-Glucuronidase
For the observation of PDP produced from an inducible promoter translating the LacZ protein from E. coli (JM109 K-12), a solution of 0.1 M pH 7 sodium phosphate buffer was used. 10 mL of this solution was used to immerse a glassy carbon electrode with a Pt mesh counter electrode and a Ag/AgCl reference. 2 mL of transformed cells (with rhamnose promoter PRha inducible uidA protein) were grown for 4 hours at 37 degrees, induced with a genetic circuit activating component (1% w/v rhamnose) for 6 hours, and then pelleted and rinsed in the phosphate buffer pH 7.0 three times. The cells were subsequently added to a tube containing 5 mM PDPG and 50 μL was added every ten minutes to the electrochemical cell for thirty minutes. At each concentration, the working electrode was then swept from −0.3 V to 0.7 V vs the Ag/AgCl reference electrode at 100 mV/s for 3 cycles and the third cycle was plotted. A voltammogram showing the results is provided in FIG. 8.
Example 8—Detection of PAP Using Inducibly Expressed β-Galactosidase
For the observation of PAP produced from an inducible promoter translating the LacZ protein from E. coli (DH5 α K-12) a solution of 0.1 M pH 7 sodium phosphate buffer was used. 10 mL of this solution was used to immerse a glassy carbon electrode with a Pt mesh counter electrode and a Ag/AgCl reference. 2 mL of transformed cells (with an IPTG inducible promoter Plac inducible lacZ protein) were grown for 4 hours at 37 degrees, induced with a genetic circuit activating component (1 mM final concentration IPTG) for 6 hours, and then pelleted and rinsed in the phosphate buffer pH 7.0 three times. The cells were subsequently added to a tube containing 5 mM PAPG and 50 μL was added every ten minutes to the electrochemical cell for thirty minutes. At each concentration, the working electrode was then swept from −0.3 V to 0.5 V vs the Ag/AgCl reference electrode at 100 mV/s for 3 cycles and the third cycle was plotted (the baseline was extended to 0.6 V to check for additional voltammogram features). A voltammogram showing the results is provided in FIG. 9.
Example 9—Simultaneous Detection of PDP and PAP Using Inducibly Expressed β-galactosidase and Inducibly Expressed β-glucuronidase
For the observation of both PDP and PAP produced from two inducible promoters translating the LacZ and UidA proteins from E. coli (Prlac and PRha promoters, respectively), a solution of 0.1 M pH 7 sodium phosphate buffer was used. 10 mL of this solution was used to immerse a glassy carbon electrode with a Pt mesh counter electrode and a Ag/AgCl reference. 2 mL of transformed cells were grown overnight (as previously described), induced with a genetic circuit activating component specific for each transformed gene (1 mM IPTG for the Plac promoter and 1% (w/v) rhamnose for the PRha promoter), and then pelleted and rinsed in the phosphate buffer. The cells were subsequently added to a tube containing 5 mM PAPG and 5 mM PDPG, and 50 μL was added every ten minutes to the electrochemical cell for thirty minutes. At each concentration, the working electrode was then swept from −0.3 V to 0.7 V vs the Ag/AgCl reference electrode at 100 mV/s for 3 cycles and the third cycle was plotted. A voltammogram showing the results is provided in FIG. 10.
Example 10—Simultaneous Detection of PDP and PAP Using Inducibly Expressed β-galactosidase and Inducibly Expressed β-glucuronidase by Differential Pulse Voltammetry
For an alternate observation of PDP and PAP produced from two inducible promoters translating the LacZ and UidA proteins from E. coli (Plac and PRha promoters, respectively), a solution of 0.1 M pH 7 sodium phosphate buffer was used. 10 mL of this solution was used to immerse a glassy carbon electrode with a Pt mesh counter electrode and a Ag/AgCl reference. 2 mL of transformed cells were grown overnight, induced with a genetic circuit activating component (1 mM IPTG for the Plac promoter and 1% (w/v) rhamnose for the PRha promoter) specific for each transformed gene, and then pelleted and rinsed in the phosphate buffer. The cells were subsequently added to a tube containing 5 mM PAPG and 5 mM PDPG, and 50 μL was added every ten minutes to the electrochemical cell for thirty minutes. At each concentration, the working electrode was held at 0.2 V vs Ag/AgCl for ten seconds and then pulsed negatively to −0.3 V vs Ag/AgCl using the differential pulse voltammetry method. The pulse height used was 5 mV for 50 ms, with a step width of 10 mV for 100 ms. A differential pulse voltammogram showing the results is provided in FIG. 11.
Example 11—Comparison of Induced Versus Non-Induced β-galactosidase
To compare the production of PAP from an inducible bacteria translating the LacZ protein from E. coli under the control of an inducible promoter, Plac, a solution of 0.1 M pH 7 sodium phosphate buffer was used. 10 mL of this solution was used to immerse a glassy carbon electrode with a Pt mesh counter electrode and a Ag/AgCl reference, 2 mL of transformed cells were grown overnight, induced with a genetic circuit activating component (1 mM IPTG) specific for the transformed gene or left non-induced, and then pelleted and rinsed in the phosphate buffer. The cells were subsequently added to a tube containing 5 mM PAPG, and 50 μL was added every ten minutes to the electrochemical cell for thirty minutes. At each concentration the working electrode was swept from −0.3 V to 0.7 V vs Ag/AgCl for three cycles. The maximum current in the oxidation peak of PAP was calculated for each sample and plotted as a function of time (occurring at approximately 0.1 V). The results are shown in FIG. 12.
Example 12—Baseline Production of PAP from E. coli Capable of Inducibly Producing β-galactosidase
For the observation of the baseline production of PAP from an inducible promoter capable of translating the LacZ protein from E. coli, under non-induced conditions, a solution of 0.1 M pH 7 sodium phosphate buffer was used. 10 mL of this solution was used to immerse a glassy carbon electrode with a Pt mesh counter electrode and a Ag/AgCl reference. 2 mL of transformed cells were grown overnight and then pelleted and rinsed in the phosphate buffer. The cells were subsequently added to a tube containing 5 mM PAPG and 50 μL was added every ten minutes to the electrochemical cell for thirty minutes. At each concentration, the working electrode was then swept from −0.3 V to 0.7 V vs the Ag/AgCl reference electrode at 100 mV/s for 3 cycles and the third cycle was plotted. The results are shown in FIG. 13.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
SUMMARY OF SEQUENCES
SEQ.ID NO:1 and 2 set forth the nucleotide sequence and deduced amino acid sequence of β-galactosidase (LacZ).
SEQ.ID NO:3 and 4 set forth the nucleotide sequence and deduced amino acid sequence of β-D-glucuronidase (UidA).
SEQ.ID NO:4 and 6 set forth the nucleotide sequence and deduced amino acid sequence of β-D-glucosidase (BglX).
SEQ.ID. NO:7 sets forth the nucleotide sequence of the lacZ promoter.
SEQ. ID. NO:8 sets forth the nucleotide sequence of the prha promoter.
SEQ. ID. NO:9 sets forth the nucleotide sequence of the tet promoter.
SEQ. ID. NO:10 sets forth the nucleotide sequence of the lambda cl promoter.
SEQ. ID. NO:11 sets forth the nucleotide sequence of the lasR promoter.
SEQ. ID. NO:12 sets forth the nucleotide sequence of the luxR promoter.
SEQ. ID. NO:13 sets forth the nucleotide sequence of the lexA promoter.
SEQ. ID. NO:14 sets forth the nucleotide sequence of the pBad/araC promoter.
SEQ. ID. NO:15 sets forth the nucleotide sequence of the fecA promoter.
SEQ. ID. NO:16 sets forth the nucleotide sequence of the cusR promoter.
SEQ. ID. NO:17 sets forth the nucleotide sequence of the T7 promoter.
SEQ. ID. NO:18 sets forth the nucleotide sequence of the σ32 promoter.
SEQ. ID. NO:19 sets forth the nucleotide sequence of the σ70 promoter.
SEQ. ID. NO: 20 sets forth the nucleotide sequence of the E. coli pRha promoter
SEQ. ID. NO: 21 sets forth the nucleotide sequence of a Thiobacillus ferrooxidans UV inducible promoter.

## Claims

1. A method for simultaneously detecting two parameters in a liquid medium comprising:
(i) providing a liquid medium comprising a first and second parameter;
(ii) contacting in a vessel the liquid medium with cells and a first and second substrate to form an assay mixture, wherein the first substrate comprises a first electroactive analyte chemically linked to a first carrier and the second substrate comprises a second electroactive analyte chemically linked to a second carrier; and wherein the cells comprise a chimeric nucleic acid construct comprising in the 5′ to 3′ direction of translation as operably linked components a first nucleic acid construct comprising:
(a) a first promoter inducible by the first parameter; and
(b) a nucleic acid sequence encoding a first reporter polypeptide comprising a first hydrolase capable of hydrolyzing the first substrate and releasing the first electroactive analyte from the carrier; and
a second nucleic acid construct comprising:
(c) a second promoter inducible by the second parameter; and
(d) a nucleic acid sequence encoding a second reporter polypeptide comprising a second hydrolase capable of hydrolyzing the second substrate and releasing the second electroactive analyte from the carrier;

(iii) detecting an electrical signal facilitated by the first and second electroactive analyte in the assay mixture, wherein detecting the electrical signal facilitated by the first and second electroactive analyte detects the first and second parameters in the liquid medium;
wherein the first and second hydrolase are independently selected from the group of hydrolases consisting of β-D-glucosidase, β-D galactosidase, and β-D-glucuronidase;
wherein the first and second substrate are independently selected from the group of substrates consisting of chlorophenol red-β-D-galactopyranoside (CPRG),para-nitrophenol-β-D-glucuronide (PNPG), para-aminophenol-β-D-galactopyranoside (PAPG) and para-di-phenol-β-D-glucopyranoside (PDPG);
wherein the voltage in step (iii) is applied to the assay mixture by a voltammetric sweep, and wherein the first and second nucleic acid constructs are different.

2. The method according to claim 1 wherein the β-D-galactosidase comprises SEQ ID NO: 2, the β-D glucuronidase comprises SEQ ID NO: 4, and the β-D-glucosidase comprises SEQ ID NO: 6.

3. A method of simultaneously detecting a first and second parameter in a liquid medium, the method comprising:
(i) contacting in a vessel the liquid medium and (a) a first substrate comprising a first electroactive analyte chemically linked to a first carrier; (b) a second substrate comprising a second electroactive analyte chemically linked to a second carrier; (c) a first reporter polypeptide comprising a first hydrolase; and (d) a second reporter polypeptide comprising a second hydrolase, to form an assay mixture, wherein the first hydrolase is capable of hydrolyzing the first substrate and releasing the first electroactive analyte from the carrier and the second hydrolase is capable of hydrolyzing the second substrate and releasing the second electroactive analyte from the carrier; and
(ii) detecting an electrical signal facilitated by the first and second electroactive analyte in the assay mixture, wherein detecting the electrical signal is facilitated by the first and second electroactive analyte detects the first and second parameters in the liquid medium;
wherein the first and second hydrolase are independently selected from the group of hydrolases consisting of β-D-glucosidase, β-D galactosidase, and β-D-glucuronidase;
wherein the first and second substrate are independently selected from the group of substrates consisting of chlorophenol red-β-D-galactopyranoside (CPRG), para-nitrophenol-β-D-glucuronide (PNPG), para-aminophenol-β-D-galactopyranoside (PAPG) and para-di-phenol-β-D-glucopyranoside (PDPG);
wherein detecting the electrical signal is performed by applying a voltage to the assay mixture by a voltammetric sweep, and wherein the first and second reporter polypeptides are different.

4. The method according to claim 3 wherein the β-D-galactosidase comprises SEQ ID NO: 2, the β-D glucuronidase comprises SEQ ID NO: 4, and the β-D-glucosidase comprises SEQ ID NO: 6.