Patent Publication Number: US-2023151354-A1

Title: Discovery and evolution of biologically active metabolites

Description:
CROSS-REFERENCE 
     This application is a continuation of International Application No.: PCT/US2021/012621, filed Jan. 8, 2021, which claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application No. 62/958,368, filed Jan. 8, 2020, each of which is incorporated by reference herein in its entirety. 
    
    
     STATEMENT AS TO FEDERALLY SPONSORED RESEARCH 
     This invention was made with government support under grant 1750244 awarded by the National Science Foundation. The government has certain rights to this invention. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on Dec. 22, 2022, is named 57123-702_301_SL.xml and is 192,400 bytes in size. 
     FIELD 
     Disclosed herein are systems, methods, reagents, apparatuses, vectors, and host cells for the discovery and evolution of metabolic pathways that produce small molecules that modulate enzyme function. 
     BACKGROUND 
     Natural products and their derivatives represent a longstanding source of pharmaceuticals and medicinal preparations 1-3 . These molecules—perhaps, as a result of their biological origin—tend to exhibit favorable pharmacological properties (e.g., bioavailability and “metabolite-likeness”) 1,4  and can exert a striking variety of therapeutic effects (e.g., analgesic, antiviral, antineoplastic, anti-inflammatory, cytotoxic, immunosuppressive, and immunostimulatory) 5-10 . Recent advances in synthetic biology and metabolic engineering have suppled new approaches for the efficient biosynthesis and functionalization of known, pharmaceutically relevant natural products 11-13 ; complementary methods for the discovery and optimization of new products with specific, therapeutically relevant activities, however, remain underdeveloped 14 . 
     Existing strategies for natural product discovery are largely undirected and/or limited in scope. For example, screens of large natural product libraries—augmented, on occasion, with combinatorial (bio)chemistry 15-17 —have uncovered molecules with important medicinal properties 18 , but these screens are resource-intensive and largely subject to serendipityl 9 . Bioinformatic tools, by contrast, permit the identification of biosynthetic gene clusters 20,21 , where co-localized resistance genes, if present, can reveal the biochemical function of their products 22 . The therapeutic activities of many pharmaceutically relevant metabolites, however, differ from their native functions 23 , and most biosynthetic pathways can, when appropriately reconfigured, yield entirely new—and, perhaps, more effective—therapeutic molecules 12,24 . 
     Microbial systems have emerged as powerful platforms for the biosynthesis of natural products from unculturable or low-yielding organisms. 25,26  Recent work showed that such systems can also permit the discovery and evolution of metabolic pathways with specific, therapeutically relevant activities (PCT/US2019/40896). 
     SUMMARY 
     Disclosed herein are systems, methods, reagents, apparatuses, vectors, and host cells for the discovery and evolution of metabolic pathways that produce small molecules that modulate enzyme function. For example, a microorganism is provided in which a first genetically encoded system links cell growth to the activity of a target enzyme and in which a second genetically encoded system—to be discovered or evolved—produces a metabolite that modulates the activity of the target enzyme. This disclosure applies this approach to a subset of target enzymes that post-translationally modify proteins, to metabolic pathways that produce phenylpropanoids or nonribosomal peptides, and to the discovery of cryptic metabolic pathways. Some aspects of this disclosure provide specific reconfigured or evolved pathways that produce specific modulators of enzyme activity, that yield improved titers of such modulators (relative to a starting pathway), and/or that exhibit reduced host toxicity (relative to a starting pathway). Metabolic products with specific inhibitory effects are also disclosed. 
     According to one aspect, methods for the discovery and evolution of metabolic pathways that produce molecules that modulate protein function are provided. The methods include contacting a population of host cells that comprise a protein of interest, such as an enzyme of interest, with a population of expression vectors comprising different metabolic pathways, wherein the host cells are amenable to transfer of the population of expression vectors; expressing the metabolic pathways in the population of host cells, wherein a cell or subset of the population of host cells produce a detectable output when the metabolic pathway within said cell or population of host cells produces a product that modulates the protein of interest, such as the enzyme of interest; screening the population of host cells under conditions that enable measurement of the detectable output in the cell or the subset of the population of host cells; isolating the cell or the subset of the population of host cells that produce a detectable output; isolating the expression vectors that yield detectable outputs higher than (p&lt;0.05) the output of a reference vector that harbors a reference pathway, for example, a vector that encodes a pathway that does not produce molecules with concentrations and/or potencies sufficient to modulate the activity of a protein of interest, such as an enzyme of interest, in the cell or the subset of the population of host cells; and characterizing the products of the metabolic pathways encoded by the expression vectors that yield detectable outputs that are higher than the output of said reference vector in the cell or the subset of the population of host cells. 
     In some embodiments, the host cells comprise a genetically encoded system in which the activity of a protein of interest, such as an enzyme of interest, controls the assembly of a protein complex with an activity that is not possessed by either of two or more components of the complex and, thus, yields a detectable output in proportion to the amount of complex formed. 
     In some embodiments, the protein of interest is an enzyme that adds a post-translational modification that causes two proteins, which are initially dissociated, to be covalently linked or to form a noncovalent complex. 
     In some embodiments, the complex is formed by two proteins with a dissociation constant (K d ) less than or equal to the K d  of the complexes formed between SH2 domains and their phosphorylated substrates. 
     In some embodiments, the enzyme of interest is an enzyme that adds a post-translational modification other than the addition or removal of a phosphate, and that modification causes two proteins, which are initially dissociated inside of the cell, to be covalently linked or to form a complex with a dissociation constant (K d ) less than or equal to the K d  of the complex formed between a SH2 domain and a phosphorylated SH2-substrate domain (e.g., as shown in  FIG.  1 A ). 
     In some embodiments, the metabolic pathways produce phenylpropanoids or nonribosomal peptides. 
     In some embodiments, the expression vectors comprising different metabolic pathways comprise a library of pathways generated by mutating one or more genes within a starting metabolic pathway. 
     In some embodiments, one or more of the metabolic pathways comprises a set of genes of unknown biosynthetic capability. 
     In some embodiments, one or more of the metabolic pathways that produces a detectable output higher than the output of the reference pathway produces a product that differs from the products of other metabolic pathways. 
     In some embodiments, one or more of the metabolic pathways that produces a detectable output higher than the output of the reference pathway produces a larger quantity of a product than the quantity of product generated by other metabolic pathways. 
     In some embodiments, one or more of the metabolic pathways that produces a detectable output higher than the output of the reference pathway exhibits a lower cellular toxicity than other metabolic pathways. 
     In some embodiments, the products of the metabolic pathways are characterized by standard analytical methods, preferably by gas chromatography-mass spectrometry (GC/MS), liquid chromatography-mass spectrometry (LC/MS), and/or nuclear magnetic resonance (NMR) spectroscopy. 
     In some embodiments, the methods further include isolating the products. 
     In some embodiments, the methods further include concentrating the products, preferably using a rotary evaporator. 
     In some embodiments, the methods further include testing the effects of the products on the protein of interest, such as the enzyme of interest. 
     In some embodiments, the protein of interest, such as the enzyme of interest, is a ubiquitin ligase, a SUMO transferase, a methyltransferase, a demethylase, an acetyltransferase, a glycosyltransferase, a palmitoyltransferase, or a related hydrolase. 
     In some embodiments, the products or molecules identified (e.g., amorphadiene and derivatives, taxadiene and derivatives, β-bisabolene and derivatives, α-bisabolene and derivatives, and α-longipinene and derivatives) are provided as drugs or drug leads for the treatment of diseases to which PTPs contribute, for example, type 2 diabetes, HER2-positive breast cancer, or Rett syndrome, as are methods of treatment of such diseases by administering an effective amount of the molecule(s) to a subject in need of such treatment. 
     According to another aspect, compositions or systems are provided that include a population of host cells that comprise a protein of interest and a population of expression vectors comprising different metabolic pathways, wherein a cell or subset of the population of host cells produce a detectable output when the metabolic pathway produces a product that modulates the protein of interest, and optionally wherein the expression vectors yield detectable outputs higher than the output of a reference vector that harbors a reference pathway, for example, a vector that encodes a pathway that does not produce molecules with concentrations and/or potencies sufficient to modulate the activity of a protein of interest, in the cell or the subset of the population of host cells. 
     In some embodiments, the host cells comprise a genetically encoded system in which the activity of a protein of interest controls the assembly of a protein complex with an activity that is not possessed by either of two or more components of the complex and, thus, yields a detectable output in proportion to the amount of complex formed. 
     In some embodiments, the protein of interest is an enzyme that adds a post-translational modification that causes two proteins, which are initially dissociated, to be covalently linked or to form a noncovalent complex. 
     In some embodiments, the complex is formed by two proteins with a dissociation constant (K d ) less than or equal to the K d  of the complexes formed between SH2 domains and their phosphorylated substrates. 
     In some embodiments, the metabolic pathways produce phenylpropanoids or nonribosomal peptides. 
     In some embodiments, the expression vectors comprising different metabolic pathways comprise a library of pathways generated by mutating one or more genes within a starting metabolic pathway. 
     In some embodiments, one or more of the metabolic pathways comprises a set of genes of unknown biosynthetic capability. 
     In some embodiments, one or more of the metabolic pathways that produces a detectable output higher than the output of the reference pathway produces a product that differs from the products of other metabolic pathways. 
     In some embodiments, one or more of the metabolic pathways that produces a detectable output higher than the output of the reference pathway produces a larger quantity of a product than the quantity of product generated by other metabolic pathways. 
     In some embodiments, one or more of the metabolic pathways that produces a detectable output higher than the output of the reference pathway exhibits a lower cellular toxicity than other metabolic pathways. 
     In some embodiments, the protein of interest is a ubiquitin ligase, a SUMO transferase, a methyltransferase, a demethylase, an acetyltransferase, a glycosyltransferase, a palmitoyltransferase, or a related hydrolase. 
     According to another aspect, kits are provided that include a population of expression vectors as described herein. In some embodiments, the kits also include the population of host cells that comprise a protein of interest as described herein. 
     Each of the limitations of the invention can encompass various embodiments of the invention. It is therefore anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIGS.  1 A- 1 E . Development of a bacterial-two hybrid system that links the inhibition of PTP1B to antibiotic resistance.  FIG.  1 A , A bacterial two-hybrid (B2H) system that detects phosphorylation-dependent protein-protein interactions. Major components include (i) a substrate domain fused to the omega subunit of RNA polymerase (yellow), (ii) an SH2 domain fused to the 434 phage cI repressor (light blue), (iii) an operator for 434cI (dark green), (iv) a binding site for RNA polymerase (purple), (v) Src kinase, and (vi) PTP1B. Src-catalyzed phosphorylation of the substrate domain enables a substrate-SH2 interaction that activates transcription of a gene of interest (GOI, black). PTP1B-catalyzed dephosphorylation of the substrate domain prevents that interaction; inhibition of PTP1B re-enables it.  FIG.  1 B , A version of the B2H system that both (i) lacks PTP1B and (ii) contains p130cas as the substrate domain and luxAB as the GOI. Inducible plasmids were used to increase expression of specific components in  E. coli ; secondary induction of Src from one such plasmid enhanced luminescence.  FIG.  1 C , A version of the B2H system that both (i) lacks PTP1B and Src and (ii) includes an SH2 domain (SH2*) with an enhanced affinity for phosphopeptides, a variable substrate domain, and LuxAB as the GOI. An inducible plasmid was used to increase expression of Src in  E. coli . Sequences for substrates p130cas (SEQ ID NO: 24), MidT (SEQ ID NO: 25), EGFR (SEQ ID NO: 27), and ShcA (SEQ ID NO: 26) are shown.  FIG.  1 D , The B2H system from c with either p130cas or MidT as substrates. A second plasmid was used to overexpress either (i) Src and PTP1B or (ii) Src and an inactive variant of PTP1B (C215S) in  E. coli . Right: Two single-plasmid B2H systems.  FIG.  1 E , The optimized system includes SH2*, the midT substrate, optimized promoters and ribosome binding sites (bb034 from  FIG.  1 D ), and SpecR as the GOI. Inactivation of PTP1B enabled a strain of  E. coli  harboring this plasmid-borne system to survive at high concentrations of spectinomycin (&gt;250 μg/ml). Error bars in  FIG.  1 B - FIG.  1 D  denote standard error with n=3 replicates. 
         FIGS.  2 A- 2 C . Biosynthesis of PTP1B-inhibiting terpenoids enables cell survival.  FIG.  2 A , A plasmid-borne pathway for terpenoid biosynthesis: (i) pMBIS, which harbors the mevalonate-dependent isoprenoid pathway of  S. cerevisiae , converts mevalonate to isopentyl pyrophosphate (IPP) and farnesyl pyrophosphate (FPP). (ii) pTS, which encodes a terpene synthase (TS) and, when necessary, a geranylgeranyl diphosphate synthase (GGPPS), converts IPP and FPP to sesquiterpenes or diterpenes.  FIG.  2 B , Four terpene synthases: amorphadiene synthase (ADS), γ-humulene synthase (GHS), abietadiene synthase (ABS), and taxadiene synthase (TXS).  FIG.  2 C , The spectinomycin resistance of strains of  E. coli  that harbor both (i) the bacterial two-hybrid (B2H) system (ii) a TS-specific terpenoid pathway (pTS includes GGPPS only when ABS or TXS are present). ADS enabled survival in the presence of high concentrations of spectinomycin. Note: ABS D404A/D621A  is catalytically inactive. B2H* contains PTP1B C215S , which is inactive. 
         FIGS.  3 A- 3 G . Strategy for microbially assisted directed evolution (MADE).  FIG.  3 A , Error-prone PCR and/or site-saturation mutagenesis of a subset of genes within a metabolic pathway yield a library of metabolic pathways.  FIG.  3 B , Microbes, each of which harbors both (i) the B2H system and (ii) a member of the pathway library, are grown in liquid culture. Note: The system shown is an  E. coli  host that harbors both (i) the B2H system and (ii) mutated terpenoid pathways (i.e., pMBIS+pTS with mutations; see  FIG.  2 A ).  FIG.  3 C , After liquid culture, the transformants are plated on solid media with different concentrations of antibiotic; hits comprise colonies that grow at antibiotic concentrations at which the wild-type pathway does not permit growth.  FIG.  3 D , The pathways of the hits are sequenced; their mutations are reintroduced into the wild-type pathway; and these reconstructed pathway variants are rescreened with drop-based plating (10 μL) on solid media with different concentrations of antibiotic. This step removes false positives (e.g., colonies that survived because of mutations located outside of the target genes).  FIG.  3 E , The confirmed hits are grown in liquid culture; their products are extracted with a hexane overlay, as needed, and concentrated in a rotary evaporator.  FIG.  3 F , GC/MS enables the identification and quantification of mutant products; NMR can assist with identification.  FIG.  3 G , Interesting metabolites (purchased or purified from culture extract) are characterized with in vitro kinetic measurements or cell studies of target modulation and/or ITC analyses of target-metabolite binding. 
         FIGS.  4 A- 4 D . Genetically encoded systems that detect metabolite-mediated modulation of post-translational modification (PTM) enzymes.  FIG.  4 A , A genetically encoded system that detects metabolite-mediated activation of enzymes E1 and/or E2. E1 adds a PTM to protein P1, allowing it to bind to P2; the newly formed P1-P2 complex activates transcription of a gene of interest (GOI, black). E2 removes the PTM from P1 and, thus, prevents complex formation. When the GOI confers a fitness advantage, inhibitors of E2 or activators of E1 enhance cell survival. When the GOI is toxic, inhibitors of E1 or activators of E2 enhance cell survival.  FIG.  4 B , An alternative detection system. E1 adds a PTM to protein P1, allowing it to bind to P2; the newly formed P1-P2 complex assembles a split protein (e.g., a fluorescent protein, a luciferase, or an enzyme that confers antibiotic resistance). E2 removes the PTM from P1 and, thus, prevents complex formation. When the reconstituted split protein confers a fitness advantage, inhibitors of E2 or activators of E1 enhance cell survival. When, by contrast, the reconstituted protein is toxic, inhibitors of E1 or activators of E2 enhance cell survival.  FIG.  4 C , A genetically encoded system that detects metabolite-mediated activation of PTM enzymes that control protein ligation (e.g., a SUMO transferase, a ubiquitin ligase, or associated peptidases). E1 attaches P1 to a lysine residue (K) of P2, and the newly formed P1-P2 complex activates transcription of a GOI. E2 breaks this complex apart.  FIG.  4 D , An alternative system. E1 attaches P1 to P2, and the newly formed P1-P2 complex permits the assembly of a split protein. E2-mediated proteolysis breaks this complex apart. 
         FIGS.  5 A- 5 C . Alternative metabolic pathways.  FIG.  5 A , Phenylpropanoid pathways developed by Young-Soo Hong and colleagues 45 . Abbreviations: TAL, ammonia-lyase from  S. espanaensis ; Sam5, 4-coumarate 3-hydroxylase form  S. espanaensis ; COM, O-methyltransferase from  A. thaliana ; ScCCL, cinnamate/4-coumarate:CoA ligase from  Streptomyces coelicolor ; CHS, chalcone synthase from  A. thaliana ; STS, stilbene synthase from  Arachis hypogaea .  FIG.  5 B , The pathways encoded by the plasmids from  FIG.  5 A .  FIG.  5 C , A genetically encodable yersiniabactin (Ybt) synthetase, as described by Khosla and colleagues 46 . Ybt is a polyketide-nonribosomal peptide. The substrates necessary for Ybt production appear in blue. Abbreviations: ArCP, aryl carrier protein; A, adenylation; PCP, peptidyl carrier proteins; Cy, cyclization; KS, ketosynthase; ACP, acyl carrier protein; AT, acyltransferase; KR, NADPH-dependent ketoreductase; MT, methyltransferase; SAM, S-adenosylmethionine; TE, thioesterase. See the text for details on biosynthesis. 
         FIGS.  6 A- 6 B . An approach for the discovery of cryptic metabolic pathways.  FIG.  6 A , Mutagenesis and/or reorganization of a multi-step pathway inactivates a biosynthetic gene and, thus, permits the accumulation of a metabolic intermediate.  FIG.  6 B , Mutagenesis and/or reorganization of a multi-step pathway inactivates a repressor gene and, thus, permits the expression of pathway genes. 
         FIGS.  7 A- 7 I . Microbial evolution of terpenoid inhibitors.  FIG.  7 A- 7 B , Homology models for ( FIG.  7 A ) ADS and ( FIG.  7 B ) GHS show the locations of residues targeted for site-saturation mutagenesis (SSM). A substrate analogue from an aligned structure of 5-epi-aristolochene synthase (pdb entry Seat) appears in blue.  FIG.  7 C- 7 D , Measurements of the spectinomycin resistance conferred by mutants of (c) ADS (LB plates) and ( FIG.  7 D ) GHS (TB plates). ALP corresponds to a quintuple mutant of GHS (A336C/T445C/S484C/I562L/M565L) that generates α-longipinene as a major product. Shades denote colony densities: diffuse 10 colonies, light gray), circular diffuse (gray), and circular lawn (black).  FIG.  7 E , The product profiles of mutants of ADS that enable growth at higher antibiotic concentrations than the wild-type enzyme.  FIG.  7 F , ADS G43S/K51N  and ADS yield similar amorphadiene titers in liquid cultures.  FIG.  7 G , ADS G43S/K51N  yields higher colony densities than the wild-type enzyme in the presence of an inactive B2H system (B2Hx); these densities suggest that ADS G43S/K51N  is less toxic than ADS.  FIG.  7 H , The product profiles of wild-type GHS and several GHS mutants that yield enhanced antibiotic resistance; discrepancies between profiles of these mutants suggest differences in the composition of intracellular terpenoids that might give rise to enhanced antibiotic resistance.  FIG.  7 I , GHS A319Q  yields a higher terpenoid titer than GHS. Error bars in  FIG.  7 F  and  FIG.  7 I  denote standard deviation with n=3 biological replicates. 
         FIGS.  8 A- 8 D . Analysis of evolved mutants.  FIG.  8 A , Analysis of the antibiotic resistance conferred by mutants of ADS. Images show the growth of  E. coli  on LB plates seeded from drops of liquid culture (10 μL). Each mutant was prepared by using site-directed mutagenesis to introduce mutations identified in the selection experiment (i.e., hits) into the starting ADS plasmid. Shades denote colony densities: diffuse (≥10 colonies, light gray), circular diffuse (gray), and circular lawn (black)  FIG.  8 B , A replicate of the experiment described in  FIG.  8 A .  FIG.  8 C , Analysis of the antibiotic resistance conferred by mutants of GHS. Images show the growth of  E. coli  on TB plates seeded from drops of liquid culture (10 μL).  FIG.  8 D , A replicate of the experiment described in  FIG.  8 C . In  FIG.  8 A - FIG.  8 D , blue highlights denote mutants that enabled growth at higher concentrations of spectinomycin than the wild-type enzymes in two biological replicates (i.e., these mutants appear in  FIGS.  3 C and  3     d ). 
         FIGS.  9 A- 9 C . Analysis of the products of different terpene synthases.  FIG.  9 A , Titers of the dominant terpenoids (i.e., amorphadiene, γ-humulene, taxadiene, or abietadiene) generated by each TS-specific strain in the absence (top) and presence (bottom) of the B2H system. Similar titers indicate that the B2H system does not interfere with terpenoid biosynthesis.  FIG.  9 B , GC/MS chromatograms of the terpenoids generated by each strain in the absence (top) and presence (bottom) of the B2H system (m/z=204). Similar profiles indicate that the B2H system does not alter product distributions.  FIG.  9 C , Analysis of the contributions of either (i) TS activity or (ii) B2H function to the death and survival of various strains. Inactivation of GHS does not enhance the survival of the GHS strain, an indication that this enzyme does not produce growth-inhibiting terpenoids. Inactivation of either ADS or the B2H system, by contrast, weakens the antibiotic resistance of the ADS strain, an indication that maximal resistance requires both terpenoid production and B2H activation. Labels denote the following controls: GHS D/A , an inactive GHS; ADS D/A , an inactive ADS; B2H*, a constitutively active B2H; B2H x , an inactive B2H. Note: The left and right images show LB plates seeded with drops of liquid culture (10 μL) from two biological replicates. Error bars in  FIG.  9 A  denote standard error for n≥3 biological replicates. 
         FIGS.  10 A- 10 E . Analysis of the products of various terpenoids.  FIG.  10 A , Chromatograms show expected dominant products (*) for each TS-specific strain from  FIG.  2 C  (the B2H system is present).  FIG.  10 B , Titers of major products generated by ADS and TXS.  FIG.  10 C , Initial rates of PTP1B-catalyzed hydrolysis of pNPP in the presence of increasing concentrations of amorphadiene and taxadiene. Lines show fits to a Michaelis-Menten model, which provides evidence of noncompetitive inhibition (amorphadiene) and mixed inhibition (taxadiene).  FIG.  10 D , A depiction of a HEK293T/17 cell. Insulin stimulates phosphorylation of the membrane-bound insulin receptor (IR); PTP1B dephosphorylates IR, and the inhibition of PTP1B restores phosphorylation.  FIG.  10 E , ELISA-based measurements of IR phosphorylation in starved wild-type HEK293T/17 cells exposed to 3% dimethyl sulfoxide (DMSO, n=2), 930 μM amorphadiene (AD, in 3% DMSO, n=3), and 405 μM α-bisabolene (Abis, 3% DMSO, n=1) for 10 minutes. The results indicate that both amorphadiene and α-bisabolene can cross the cell membrane, inhibit intracellular PTP1B, and, thus, increase IR phosphorylation. Error bars in  FIG.  10 B  denote standard error with n=3 biological replicates. Error bars in  FIG.  10 C  denote standard error with n≥3 measurements. Error bars in  FIG.  10 E  denote standard error with n values indicated (we note: for these measurements, we subtracted a reference signal produced by lysis buffer alone, n=3). 
         FIGS.  11 A- 11     d . Analysis of alternative terpene synthases.  FIG.  11 A - FIG.  11 B , The spectinomycin resistance of strains of  E. coli  that harbor (i) an active or inactive bacterial two-hybrid system (B2H and B2Hx, respectively, as in  FIGS.  1 ,  2 , and  7 - 9   ) and (ii) the terpenoid pathway from  FIG.  2    with each of the following terpene synthases: γ-humulene synthase from  Abies grandis  (GHS), β-bisabolene synthase from  Zingiber officinale  (ZoBBA), β-bisabolene synthase from  Santalum album  (SaBBA), and α-bisabolene synthase (ABB) from  Abies grandis  (ABS). SaBBA and, most prominently, ABB enable survival at high concentrations of spectinomycin.  FIG.  11 C , chemical structures of β-bisabolene and α-bisabolene.  FIG.  11 D , analysis of PTP1B activity on p-nitrophenyl phosphate (pNPP) in the presence of increasing concentrations of α-bisabolene (measured as amorphadiene equivalents) purified from culture extract. Lines show fits to a Michaelis-Menten Model. 
         FIGS.  12 A- 12 G . Analysis of selective inhibitors of PTP1B.  FIG.  12 A , Initial rates of pNPP hydrolysis by PTP1B 321 , TCPTP 292 , and PTP1B 282  in the presence of increasing concentrations of amorphadiene. Lines show fits to models of inhibition. A comparison of the first and second plots (or, more specifically, the IC 50 &#39;s derived from the plotted data) indicates that amorphadiene is a ˜five-fold more potent inhibitor of PTP1B 321  than TCPTP 292 , the most closely related PTP in the human genome (by sequence identity); this selectivity suggests that amorphadiene binds outside of the active site of PTP1B. A comparison of the second and third plots, in turn, indicate that amorphadiene inhibits PTP1B 282  ˜four-fold less potently than PTP1B 321 ; this discrepancy suggests that the α7 helix, which is present in PTP1B 321  but missing in PTP1B 282  (and which is proximal to a known allosteric binding site of PTP1B), is involved in the PTP1B 321 -amorphadiene interaction.  FIG.  12 B , the chemical structure of amorphadiene.  FIG.  12 C , a preliminary crystal structure of PTP1B bound to amorphadiene.  FIG.  12 D , Data used to solve the structure in  FIG.  12 C  shows electron density near the allosteric site of PTP1B (F280 appears on the left of this image); this density is consistent with the structure of amorphadiene.  FIG.  12 E , the chemical structure of α-bisabolol, a structural analogue of α-bisabolene.  FIG.  12 F , a preliminary crystal structure of PTP1B bound to α-bisabolol.  FIG.  12 G , Data used to solve the structure in  FIG.  12 F  shows electron density near the allosteric site of PTP1B (F280 appears in the upper left of this image); this density is consistent with the structure of α-bisabolol. 
         FIG.  13   . Optimization of the bacterial-two hybrid (B2H) system.  FIG.  13   , We optimized the transcriptional response of the B2H system by adjusting the strength of various genetic elements. In three sequential phases, we changed (1) the promoter for Src/CDC37, (2) the ribosome binding site (RBS) for Src/CDC37, and (3) and the RBS for PTP1B. In phases 1 and 2, we used a PTP1B-deficient system with either a wild-type (WT, EPQ Y EEIPYL (SEQ ID NO:1)) or non-phosphorylatable (Mut, EPQ F EEIPYL (SEQ ID NO:2)) substrate domain. Here, “none” indicates that absence of an additional promoter; the labeled “Prol” controls the transcription of all five genes to its left. In phase 3, we used a complete B2H system with either a wild-type (WT) or catalytically inactive (C215S, Mut) variant of PTP1B. The remaining B2H component of each phase are detailed in TABLE 2. Error bars denote standard error with n≥3 biological replicates. 
         FIG.  14   . Analysis of different selection conditions.  FIG.  14   , A comparison of the antibiotic resistance conferred by B2H systems with different RBSs for PTP1B (see TABLE 2 for the remaining components of each system). Images show the growth of  E. coli  on agar plates (LB) seeded from drops of liquid culture (10 μL) with two biological replicates for each condition. The RBS bb034 confers a greater sensitivity to spectinomycin on agar plates; concentrations of spectinomycin in the liquid culture, by contrast, do not have a strong influence on bacterial growth. Informed by this analysis, we incorporated bb034 into our “optimized” B2H system and ceased adding spectinomycin to liquid culture. 
         FIGS.  15 A- 15 B .  FIG.  15 A , A GC chromatogram of pure amorphadiene (purchased from Ambeed).  FIG.  15 B , The mass spectrum of the indicated peak from  FIG.  15 A . 
         FIGS.  16 A- 16 B . GC/MS analysis of  -humulene production.  FIG.  16 A , A GC chromatogram shows the production of  -humulene by a strain of  E. coli  engineered to produce it (i.e., pMBIS+pGHS).  FIG.  16 B , The mass spectrum of the indicated peak from  FIG.  16 A . 
         FIGS.  17 A- 17 B . Supplementary  FIG.  4   |GC/MS analysis of abietadiene production.  FIG.  17 A , A GC chromatogram shows the production of abietadiene by a strain of  E. coli  engineered to produce it (i.e., pMBIS+pABS).  FIG.  17 B , The mass spectrum of the indicated peak from  FIG.  17 A . 
         FIGS.  18 A- 18 B . GC/MS analysis of taxadiene production.  FIG.  18 A , A GC chromatogram shows the production of pure taxadiene (a kind gift from Phil Baran).  FIG.  18 B , The mass spectrum of the indicated peak from  FIG.  18 A . 
         FIGS.  19 A- 19 B . GC/MS analysis of β-bisabolene production.  FIG.  19 A , A GC chromatogram shows the production of β-bisabolene by a strain of  E. coli  engineered to produce it (i.e., pMBIS+pGHS L450G ).  FIG.  19 B , The mass spectrum of the indicated peak from  FIG.  19 A . 
         FIG.  20   . Standard curve for pNPP assay. This standard curve was generated by dissolving various concentrations of p-nitrophenol (p-NP) in 100 μL water and measuring their absorbance with a plate reader. Absorbance measurements collected in our pNPP kinetics analysis were converted to concentrations using this curve. 
         FIGS.  21 A- 21 E . Development of a bacterial-two hybrid system that links the inhibition of PTP1B to antibiotic resistance. This figure elaborates on  FIG.  1    by including the orientation of genes.  FIG.  21 A , A bacterial two-hybrid (B2H) system in which a phosphorylation-dependent protein-protein interaction modulates transcription of a gene of interest (GOI, black). Major components include (i) a substrate domain fused to the omega subunit of RNA polymerase (yellow), (ii) an SH2 domain fused to the 434 phage cI repressor (light blue), (iii) Src kinase and PTP1B, (iv) an operator for 434cI (dark green), (v) a binding site for RNA polymerase (purple), and (vi) a gene of interest (GOI, black).  FIG.  21 B , The luminescence generated by a B2H system with a p130cas substrate, LuxAB as the GOI, and no PTP1B. We used an inducible plasmid to increase expression of specific components. 
         FIG.  21 C , The luminescence generated by B2H systems with an SH2 domain that exhibits enhanced affinity for phosphopeptides (SH2*), one of four substrate domains, LuxAB as the GOI, and no Src or PTP1B. We used an inducible plasmid to control the expression of Src. Sequences for substrates p130cas (SEQ ID NO: 24), MidT (SEQ ID NO: 25), EGFR (SEQ ID NO: 27), and ShcA (SEQ ID NO: 26) are shown.  FIG.  21 D , The B2H system from c with either p130cas or MidT substrates. We used a second plasmid to control the expression of Src and an active or inactive (C215) variant of PTP1B. Right: Two optimized single-plasmid systems.  FIG.  21 E , The final B2H system. Inactivation of PTP1B enabled a strain of  E. coli  harboring this system to survive at high concentrations of spectinomycin (&gt;250 μg/ml). Error bars in  FIGS.  21 B- 21 D  denote standard error with n=3 biological replicates. 
         FIGS.  22 A- 22 G . Biosynthesis of PTP1B-inhibiting terpenoids enables cell survival. This figure elaborates on  FIGS.  2  and  10   .  FIG.  22 A , The plasmid-borne pathway for terpenoid biosynthesis: (i) pMBIS CmR , which harbors the mevalonate-dependent isoprenoid pathway of  S. cerevisiae , converts mevalonate to isopentyl pyrophosphate (IPP) and farnesyl pyrophosphate (FPP). (ii) pTS, which encodes a terpene synthase (TS) and, when necessary, a geranylgeranyl diphosphate synthase (GGPPS), converts IPP and FPP to sesquiterpenes or diterpenes.  FIG.  22 B , Five terpene synthases examined in this study: amorphadiene synthase (ADS), γ-humulene synthase (GHS), α-bisabolene synthase (ABA), abietadiene synthase (ABS), and taxadiene synthase (TXS).  FIG.  22 C , The spectinomycin resistance of strains of  E. coli  that harbor both (i) the bacterial two-hybrid (B2H) system (ii) a TS-specific terpenoid pathway. Note: ABS*, a positive control, has a constitutively active B2H (i.e., it includes PTP1B C215S ).  FIG.  22 D , Chromatograms show expected major products (i.e., namesake; *) for each TS-specific strain from c in the presence of the B2H system. Values are normalized to the largest peak within a given sample.  FIG.  22 E , Initial rates of PTP1B-catalyzed hydrolysis of pNPP in the presence of increasing concentrations of (AD) amorphadiene or (AB) α-bisabolene. Lines show the best-fit kinetic models of inhibition (TABLE 12).  FIG.  22 F , Estimated IC 50 &#39;s.  FIG.  22 G , Titers of the major products generated by ADS and ABA. Error bars denote ( FIG.  22 E ) standard error and ( FIG.  22 F ) 95% confidence intervals for n≥3 independent measurements, and ( FIG.  22 G ) standard deviation for n=3 biological replicates. 
         FIGS.  23 A- 23 H . Biophysical analysis of terpenoid-mediated inhibition. This figure builds on  FIG.  12    by including additional kinetic measurements.  FIG.  23 A . Aligned X-ray crystal structures of PTP1B bound to TCS401, a competitive inhibitor (yellow protein, orange highlights, and green spheres; pdb entry 5k9w), and BBR, an allosteric inhibitor (gray protein, blue highlights, and light blue spheres; pdb entry 1t4j).  FIG.  23 B , Aligned structures of PTP1B bound to BBR (white protein and light blue ligand) and amorphadiene (cyan protein and dark blue ligand, pdb entry 6W30).  FIG.  23 C , Dihydroartemisinic acid (DHA), a structural analogue of amorphadiene with a carboxyl group likely to disrupt binding to the hydrophobic cleft.  FIG.  23 D , DHA is eight-fold less potent than amorphadiene. Lines show the best-fit kinetic models of inhibition (TABLE 12). Error bars denote standard error for n=3 independent measurements with a 95% confidence interval for the IC 50 .  FIG.  23 E , Dixon plot showing V o   −1  vs. [TCS401] at various concentrations of AD (black, blue, purple markers). The parallel lines indicate that TCS401 and AD cannot bind simultaneously.  FIG.  23 F , Dixon plot showing V o   −1  vs. [orthovanadate] at various concentrations of AD (black, blue, purple markers). The intersecting lines indicate that orthovanadate and AD can bind simultaneously.  FIG.  23 G , Both amorphadiene and α-bisabolene inhibit PTP1B much more potently than TC-PTP; the removal of the α7 helix (or equivalent) from both enzymes reduces the selectivity of AD, but not AB. Error bars show propagated 95% confidence intervals estimated from n≥3 independent measurements at each condition.  FIG.  23 H , Amorphadiene (930 μM) and α-bisabolene (405 μM) stimulate IR phosphorylation in HEK293T/17 cells; at the same concentrations, dihydroartemisinic acid (DHA) and α-bisabolol (ABOL) exhibit reduced signals consistent with their reduced potencies (#: p&lt;0.05, compared to negative control,*: p&lt;0.05). All inhibitors are dissolved in 3% DMSO (v/v; negative control). Error bars in  FIGS.  23 D-f  denote standard error for n=3-12 biological replicates. Error bars in  FIG.  23 G  denote propagated 95% confidence intervals for n≥3 independent measurements. Error bars in  FIG.  23 H  denote standard error propagated from a buffer-only control (n=3 biological replicates). 
         FIGS.  24 A- 24 E . Analysis of uncharacterized terpene synthase genes.  FIG.  24 A , A bioinformatic analysis of terpene synthases. We assembled a cladogram of 4,464 members of the largest terpene synthase family (PF03936) and annotated it with functional data. We selected three genes from each of eight clades (curved boxes): six with no characterized genes (i.e., genes with known functions) and two with no characterized genes.  FIG.  24 B , The spectinomycin resistance conferred by the selected genes alongside pMBIS CmR  and pB2H opt . Hits with robust growth beyond 400 ug/mL spectinomycin appear in blue. “n.m.” indicates the condition was not measured.  FIG.  24 C , A0A0C9VSL7 produces (+)-1(10),4-cadinadiene as a dominant product (m/z=204).  FIG.  24 D , Structure of (+)-1(10),4-cadinadiene.  FIG.  24 E , The inhibition of PTP1B by (+)-1(10),4-cadinadiene (85% purity, 10% DMSO). Lines show the best-fit kinetic models of inhibition (TABLE 12). 
         FIGS.  25 A- 25 C .| Extension to other disease-related PTPs.  FIG.  25 A , The spectinomycin resistance of strains harboring B2H systems modified to detect the inactivation of different disease-relevant PTPs. Inactivating mutations 86-88  confer survival at high concentrations of antibiotic.  FIG.  25 B , A comparison of the resistance conferred by PTP1B- and TC-PTP-specific B2H systems in the presence of metabolic pathways for amorphadiene and α-bisabolene (i.e., pMBIS CmR +ADS or ABA). The PTP1B-specific system exhibits a prominent survival advantage, a finding consistent with the selectivity of both terpenoids for this enzyme.  FIG.  25 C , The titers of AD and AB in strains harboring both the B2H systems and associated metabolic pathways are indistinguishable between strains. 
         FIG.  26 A- 26 D . Analysis of the products of different terpene synthases. This figure builds on  FIG.  9    by including additional measurements.  FIG.  26 A , Total terpene titers generated by each TS-specific strain in the absence (red) and presence (blue) of the B2H system. These results indicate that the B2H system does not disrupt terpenoid biosynthesis.  FIG.  26 B , GC/MS chromatograms of the terpenoids generated by the diterpene synthases in the absence (top) and presence (bottom) of the B2H system (m/z=272).  FIG.  26 C , GC/MS chromatograms of the terpenoids generated by the sesquiterpene synthases in the absence (top) and presence (bottom) of the B2H system (m/z=204). Similar profiles in  FIG.  26 B  and  FIG.  26 C  indicate that the B2H system does not alter product distributions.  FIG.  26 D , Analysis of the contributions of either (i) TS activity or (ii) B2H function to the death and survival of GHS, ADS, and ABA strains. Inactivation of GHS does not enhance survival, an indication that this enzyme does not produce growth-inhibiting terpenoids. Inactivation of either ADS, ABA, or the B2H system, by contrast, weakens the antibiotic resistance of the ADS and ABA strains; maximal resistance thus requires both terpenoid production and B2H activation. Labels denote the following controls: D/A, an inactive terpene synthase (contains a D/A mutation at the catalytic aspartic acid, preventing the initial metal-binding step in terpene cyclization); *, a constitutively active B2H (contains PTP1B C215S , preventing dephosphorylation); X, an inactive B2H (contains a substrate domain with a Y/F mutation, prohibiting phosphorylation and thus binding with the SH2 domain). Images show LB plates seeded with drops of liquid culture (10 μL) from two biological replicates. TABLE 2 details the B2H systems used for these analyses. Error bars in  FIG.  26 A  denote standard deviation for n≥3 biological replicates. 
         FIG.  27   . An annotated cladogram of terpene synthases. This cladogram of the PF03936 family is surrounded by a heatmap that shows the presence/absence of known EC numbers of the form 4.2.3.# (which includes terpene cyclization reactions) from the Uniprot database. We selected three genes from each of eight clades: six with no characterized genes (red) and two with characterized genes (blue). TABLE 1 summarizes the genes. 
         FIG.  28   . Analysis of selected genes. We searched for sesquiterpene inhibitors of PTP1B by screening each of the 24 uncharacterized genes alongside the FPP pathway (i.e., pMBIS). These pictures show the antibiotic resistance conferred by each gene. We selected strains with antibiotic resistance exceeding 400 μg/ml as hits (blue). Importantly, for these genes, the reduced survival of B2Hx controls indicates that enhanced resistance requires activation of the B2H system. In the top diagrams, n.m. indicates conditions that were not measured. 
         FIG.  29   . Product profiles of selected hits. The product profiles of selected hits (extracted ion chromatograms, m/z=204). In brief, we grew up hits (i.e., pB2H opt , pMBIS CmR , and pTS) in liquid culture for 72 hours. With the exception of A0A0G2ZSL3, all hits were grown in 10 mL of 2% TB; A0A0G2ZSL3 was grown in a 4-mL culture of 2% TB. Notably, both A0A0C9VSL7 and A0A2H3DKU3 generate one dominant product: (+)-1(10),4-cadinadiene and β-farnesene, respectively. We focused on A0A0C9VSL7 because (+)-1(10),4-cadinadiene is a structural analog of amorphadiene, an inhibitor identified in our initial screen. 
         FIG.  30   . Crystallographic analysis of PTP1B bound to AD. Crystal structures of PTP1B collected in the (left) presence or (right) absence of AD. Resolutions: 2.10 Å (PTP1B-AD) and 1.94 Å (PTP1B). We refined these structures by modeling (top) the PTP1B-AD complex or (bottom) the apo form PTP1B. For PTP1B soaked with AD (left), the 1.0 σ 2Fo-Fc electron density supports the modeled position of AD but suggest multiple conformations; this density appears even when AD is excluded from the model. For apo PTP1B (right), the 1.0 σ 2Fo-Fc electron does not support a bound AD molecule; small regions of unexplained density may reflect water molecules or partial occupancy of the α7 helix 15 . 
         FIG.  31   . Crystallographic analysis of PTP1B bound to ABol. Crystal structures of PTP1B collected in the (left) presence or (right) absence of ABol. Resolutions: 2.11 Å (PTP1B-ABol) and 1.94 Å (PTP1B). We refined these structures by modeling (top) the PTP1B-ABol complex or (middle/bottom) the apo form PTP1B. For PTP1B soaked with ABol (left), the 0.90 σ 2Fo-Fc electron density is consistent with the modeled position of ABol, but it becomes less pronounced when ABol is excluded from the model. The apo form of PTP1B (right) shows similar density for both models; small differences in the shape of the 0.90 σ 2Fo-Fc electron density between datasets suggests that this density may have a different origin (e.g., a ligand vs. partial occupancy of the α7 helix). The unambiguous determination of a binding site for α-bisabolol requires additional data. 
         FIGS.  32 A- 32 C . Evidence of multiple bound conformations.  FIG.  32 A , Snapshots from molecular dynamics (MD) simulations of PTP1B bound to amorphadiene (AD). Arrows indicate clusters of ligand.  FIG.  32 B , A crystal structure of PTP1B bound to AD highlights residues that undergo high-frequency contacts. Here, contacts have residue-ligand distances &lt;4 Å, and high frequencies exceed 10% of all snapshots in the MD simulations.  FIG.  32 C , Estimates of the average root-mean-square deviation (RMSD) of the complete system (PL), the protein (P), the protein core (P core ; residues 1-287), the disordered region of the protein (P tail ; residues 288-321), and the ligand (L) over MD simulations indicate that both AD and the disordered region of the protein are mobile (the latter more so than the former), while the protein core remains fixed. The average RMSDs of both (i) the re-centered ligand (Int), a metric for rotational and vibrational fluctuations, and (ii) the center of mass (COM) of the ligand, a metric for its positional deviation, are large, an indication that the ligand can adopt multiple bound conformations and/or positions. 
         FIGS.  33 A- 33 M . Summary of kinetics analyses.  FIG.  33 A , Aligned crystal structures of PTP1B (gray, pdb entry 5k9w) and TC-PTP (blue, pdb entry 118k). Highlights on PTP1B: a competitive inhibitor (orange), the α7 helix (red), and truncation points used for kinetic studies (281 and 283, the 281-equivalent of TC-PTP).  FIG.  33 B , Sequence alignment of the α6/7 regions of PTP1B (SEQ ID NO: 140) and TC-PTP (SEQ ID NO: 141). The truncation points used in our kinetics analysis.  FIG.  33 C , aligned structures of the binding sites of BBR (gray, pdb entry 1t4j) and amorphadiene (blue).  FIG.  33 D - FIG.  33 M , Initial rates of pNPP hydrolysis by various PTPs in the presence of increasing concentrations of ( FIG.  33 D - FIG.  33 G ) amorphadiene, ( FIG.  33 H - FIG.  33 K ) α-bisabolene, ( FIG.  33 L ) dihydroartimesinic acid, and ( FIG.  33 M ) α-bisabolol inhibition. In all figures, lines show the best-fit models of inhibition (TABLE 12). Error bars in  FIG.  33 D - FIG.  33 M  represent standard error of at least 3 measurements. Error in IC 50 &#39;s represent 95% confidence intervals determined from fits to models of inhibition (TABLE 12). 
         FIGS.  34 A- 34 D . Expanded analysis of selectivity.  FIG.  34 A , Initial rate data for AD inhibition of SHP1. The lower panel shows the same data as % inhibition for a subset of points at two different substrate concentrations (open vs. closed circles).  FIG.  34 B , Initial rate data for AD inhibition of SHP2. The lower panel shows the same data as % inhibition for a subset of points at two different substrate concentrations (open vs. closed circles).  FIG.  34 C , Initial rate data for AB inhibition of SHP1. The lower panel shows the same data as % inhibition for a subset of points at two different substrate concentrations (open vs. closed circles).  FIG.  34 D , Initial rate data for AB inhibition of SHP2. The lower panel shows the same data as % inhibition for a subset of points at two different substrate concentrations (open vs. closed circles). In  FIG.  34 A ,  FIG.  34 C , and  FIG.  34 D , our inability to measure inhibition &gt;25% (lower panel) at the solubility limit of AD, in combination with the high K m  for 4-methylumbelliferyl phosphate (4-MUP), precluded accurate inhibition model fitting, K I , and IC 50  determination. However, the weak inhibition observed suggests AD/AB are less potent inhibitors of these enzymes than PTP1B. In all panels, error bars denote standard error of n=3 biological replicates and lines show fit to a noncompetitive inhibition model. 
         FIG.  35 A- 35 C . Analysis of PTP1B-mediated IR dephosphorylation.  FIG.  35 A , A depiction of insulin signaling in HEK293T/17 cells. Extracellular insulin binds to the transmembrane insulin receptor (IR), triggering phosphorylation of its intracellular domain. PTP1B, which localizes to the endoplasmic reticulum (ER) of mammalian cells, dephosphorylates this domain to regulate downstream signaling pathways. In starved cells, exogenously supplied inhibitors can permeate the cell membrane and inhibit PTP1B-mediated dephosphorylation of the IR.  FIG.  35 B , A screen of inhibitor concentrations for enzyme-linked immunosorbent assay (ELISAs). An enzyme-linked immunosorbent assay (ELISA) of IR phosphorylation in HEK293T/17 cells incubated with various concentrations of amorphadiene, α-bisabolene, and their structural analogues. We used this screen to identify biologically active concentrations of amorphadiene and α-bisabolene to study further.  FIG.  35 C , ELISA-based measurements of IR phosphorylation in HEK293T/17 cells incubated with amorphadiene (AD), α-bisabolene (AB), dihydroartimesnic acid (DHA), and α-bisabolol (ABOL). Curves denote fits to the four-parameter logistic equation: y=d+(a−d)/(1+(x/c){circumflex over ( )}b), where y is absorbance at 450 nm, and x is the sample dilution (e.g., 1 denotes no dilution, 0.5 denotes a 2-fold dilution, and so on). These signals indicate that amorphadiene and α-bisabolene can increase IR phosphorylation over a negative control (3% DMSO) and their less inhibitory analogs. Error bars denote standard error with n≥3 biological replicates. 
         FIGS.  36 A- 36 C . Full datasets for B2H-mediated antibiotic resistance.  FIG.  36 A , Biological replicates for  FIG.  22 C .  FIG.  36 B , Biological replicates for  FIG.  25 A .  FIG.  36 C , Biological replicates for  FIG.  25 B . Orange highlights correspond to the data displayed in  FIGS.  2 C and  5 A- 5 B . 
         FIGS.  37 A- 37 B . GC/MS analysis of α-bisabolene production.  FIG.  37 A , A GC/MS chromatogram shows the production of α-bisabolene by a strain of  E. coli  engineered to produce it (i.e., pMBIS+pABA).  FIG.  37 B , The mass spectrum of the indicated peak from  FIG.  37 A . 
         FIGS.  38 A- 38 B . Supplementary  FIG.  20   |GC/MS analysis of (+)-1(10),4-Cadinadiene.  FIG.  38 A , A GC/MS chromatogram shows the production of (+)-1(10),4-Cadinadiene by a strain of  E. coli  engineered to produce it (i.e., pMBIS+pA0A0C9VSL7).  FIG.  38 B , The mass spectrum of the indicated peak from  FIG.  38 A . 
         FIGS.  39 A- 39 B . A standard curve for p-nitrophenol (p-NP). This figure elaborates on  FIG.  20    by including additional measurements.  FIG.  39 A , We dissolved different amounts of p-nitrophenol (p-NP) in 100 μL buffer (50 mM HEPES, pH=7.3) and measured the absorbance of the resulting solutions with a SpectraMax M2 plate reader. A linear fit to this curve allowed us to convert absorbance measurements taken during kinetic assays (pNPP) to p-NP concentrations.  FIG.  39 B , We dissolved different amounts of 4-methyl umbelliferone (4-MU) in 100 μL buffer (50 mM HEPES, pH=7.3) and measured the FLUORESCECE of the resulting solutions with a SpectraMax M2 plate reader. A linear fit to this curve allowed us to convert absorbance measurements taken during kinetic assays (4-MUP) to 4-MU concentrations. 
     
    
    
     DETAILED DESCRIPTION 
       E. coli  is a valuable platform for the production of terpenoids 27-29 . The inventors hypothesized that a strain of  E. coli  programmed to detect the inactivation of a human drug target might enable the rapid discovery and biosynthesis of terpenoids that inhibit that target. To program such a strain, a bacterial two-hybrid (B2H) system was assembled in which a protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) from  H. sapiens  control gene expression. PTKs are targets of over 30 FDA-approved drugs 30 ; PTPs lack clinically approved inhibitors but contribute to an enormous number of diseases 31,32 . The first proof-of-concept system was specifically designed to detect inhibitors of protein tyrosine phosphatase 1B (PTP1B), an elusive therapeutic target for the treatment of type 2 diabetes, obesity, and breast cancer ( FIG.  1 A ) 31-35 . In this system, Src kinase phosphorylates a substrate domain, enabling a protein-protein interaction that activates transcription of a gene of interest (GOI). PTP1B dephosphorylates the substrate domain, preventing that interaction, and the inactivation of PTP1B re-enables it.  E. coli  is a particularly good host for this detection system because its proteome is sufficiently orthogonal to the proteome of  H. sapiens  to minimize off-target growth defects that can result from the regulatory activities of Src and PTP1B 36 . 
     B2H development was carried out in several steps. To begin, a luminescent “base” system was assembled in which Src modulates the binding of a substrate domain to a substrate homology 2 (SH2) domain; this system was based on a previous design in which protein-protein association controls GOI expression 37 . The initial system did not yield a phosphorylation-dependent transcriptional response, however, so it was complemented with inducible plasmids—each harboring a different system component—to identify proteins that might exhibit suboptimal activities. Notably, secondary induction of Src increased luminescence, an indication that insufficient substrate phosphorylation depressed GOI expression in the base system ( FIG.  1 B ). Accordingly, this system was modified by swapping in different substrate domains, by adding mutations to the SH2 domain that enhance its affinity for phosphopeptides 38 , and by removing the gene for Src. With this configuration, induction of Src from a second plasmid increased luminescence most prominently for the MidT substrate ( FIG.  1 C ); simultaneous induction of both Src and PTP1B, in turn, prevented that increase ( FIG.  1 D ). The MidT system was finalized by integrating genes for Src and PTP1B, by adjusting promoters and ribosome binding sites to amplify its transcriptional response further ( FIGS.  1 D,  13 , and  14   ), and by adding a gene for spectinomcyin resistance (SpecR) as the GOI. The final plasmid-borne detection system required the inactivation of PTP1B to permit growth at high antibiotic concentrations ( FIG.  1 E ). 
     The B2H system was used to identify new inhibitors of PTP1B by coupling it with metabolic pathways that might generate such molecules in  E. coli . Previous screens of plant extracts have identified structurally complex terpenoids that inhibit PTP1B 39 ; pathways were, thus, constructed for several simpler terpenoid scaffolds that lack established inhibitory effects: amorphadiene, γ-humulene, abietadiene, and taxadiene. Abietadiene is a metabolic precursor to a weak inhibitor of PTP1B 40 ; the other three terpenoids represent a structurally diverse set of molecules. Each pathway consisted of two plasmid-borne modules ( FIG.  2 A ): (i) the mevalonate-dependent isoprenoid pathway from  S. cerevisiae   41  and (ii) a terpene synthase supplemented—when necessary for diterpenoid production—with a geranylgeranyl diphosphate synthase. These modules enabled terpenoid titers of 0.5-100 μM in  E. coli  ( FIG.  9   ). 
     Each pathway was screened for its ability to produce inhibitors of PTP1B by transforming  E. coli  with plasmids harboring both the pathway of interest and the B2H system. GC-MS traces confirmed that all pathways generated terpenoids in the presence of the B2H system ( FIG.  2 D ). Surprisingly, the amorphadiene pathway permitted survival at high concentrations of antibiotic; importantly, maximal resistance required a functional B2H system ( FIG.  9 C ). This result suggests that the amorphadiene pathway produces an inhibitor of PTP1B. 
     Microbially-assisted directed evolution (MADE) refers to the approach described herein for using microbial systems to discover and evolve metabolic pathways that produce inhibitors or activators of a therapeutically relevant enzyme target, wherein both the metabolic pathway and the target enzyme exist within a host cell, for example, an  E. coli  cell ( FIG.  3   ). Some aspects of this approach provide a method for building a genetically encoded system that detects the activity of a target enzyme within a host cell, for example a system that links changes in the activity of a target enzyme to changes in the antibiotic resistance of the host cell ( FIG.  1   ). 
     Previous work demonstrated (i) the assembly of a detection system that links the activities of a protein kinase and a protein phosphatase to antibiotic resistance ( FIG.  1   ) and (ii) the use of that system, in combination with MADE, to discover inhibitors of a protein phosphatase ( FIG.  2   ). These results are detailed in PCT/US2019/40896. 
     Described herein are strategies, systems, methods, and reagents to expand the scope of capabilities of MADE and to address the needs of previously described evolution experiments. The MADE methods herein utilize one or more of the following: 1) target enzymes that post-translationally modify proteins (PTM enzymes) in a manner other than adding or removing a phosphate group; 2) a metabolic pathway that generates phenylpropanoids or nonribosomal peptides; 3) a cryptic gene cluster that encodes putative natural products; and 4) natural products with specific inhibitory effects. 
     In some embodiments, provided are methods for using MADE to discover and evolve metabolic pathways that produce inhibitors or activators of PTM enzymes ( FIG.  3   ), wherein said PTM enzymes modulate a protein-protein interaction that controls a detectable output, wherein both the PTM enzymes and the detectable output are encoded by at least one plasmid or one genome, wherein a metabolic pathway that produces natural products is encoded by at least one plasmid or one genome, and wherein said plasmids and genomes exist within the same host cell. In some embodiments, a pool of said host cells, each of which contains a different metabolic pathway, is screened for a detectable output, and the cells that yield the highest detectable output are selected as hits. These hits are analyzed with the following steps: 1) their metabolic pathways are reassembled from a starting pathway; 2) the reassembled pathways are re-screened in host cells (a confirmation step); 3) the cells that yield the highest detectable outputs are, once again, selected as hits; 4) these selected cells are grown in liquid culture; 5) the products generated in said liquid culture are identified and quantified with standard analytical methods, for example, gas chromatography-mass spectrometry (GC/MS); 6) the products generated in liquid culture are concentrated with a rotary evaporator; and 7) the modulatory effects of the concentrated products are tested on purified PTM enzymes ( FIG.  3   ). 
     In some embodiments, the target PTM enzyme naturally inhibits the growth of a host cell, for example, an  S. cerevisiae  cell in which a heterologously expressed kinase slows cell growth. 
     In some embodiments, the PTM enzymes are ubiquitin ligases, SUMO transferases, methyltransferases, demethylases, acetyltransferases, glycosyltransferases, palmitoyltransferases, and/or related hydrolases. In some embodiments, a bacterial two-hybrid (B2H) system links the activity of one or more PTM enzymes to the transcription of a gene of interest (GOI;  FIG.  4 A ). In some embodiments, the PTM enzymes modulate the assembly of a split protein, for example, a fluorescent protein, a luciferase, or an enzyme that confers antibiotic resistance ( FIG.  4 B ). In some embodiments, the target enzymes covalently link or proteolyze two proteins, wherein the assembly of these proteins activates the transcription of a gene of interest ( FIG.  4 C ) or reassembles a split protein ( FIG.  4 D ). 
     In some embodiments, provided are methods for the discovery and evolution of phenylpropanoids or nonribosomal peptides that inhibit or activate a target enzyme, wherein a metabolic pathway that produces phenylpropanoids or nonribosomal peptides is encoded by at least one plasmid or one genome ( FIG.  5   ), wherein said plasmid and said genome exist within a host cell, wherein mutagenesis and/or modulation of said metabolic pathways permit the production of an inhibitor or activator of the target enzyme, and wherein MADE enables the identification of pathways thus mutated and/or reconfigured. 
     In some embodiments, provided are methods for the discovery and evolution of cryptic metabolic pathways that generate inhibitors or activators of a target enzyme, wherein said cryptic metabolic pathways comprise a set of genes with unknown or poorly characterized products, or wherein said cryptic metabolic pathways comprise a set of genes in which one gene hinders the biosynthesis of an important product, wherein subsequent mutagenesis and/or reconfiguration of said pathway causes it to generate more of that product, and wherein MADE enables the discovery of a pathway thus mutated and/or reconfigured. For example, the removal of a biosynthetic gene may enable the accumulation of a metabolic intermediate that modulates the activity of a target enzyme ( FIG.  6 A ); alternatively, the removal of a gene for a transcriptional repressor may permit the activation of the entire metabolic pathway ( FIG.  6 B ). 
     In some embodiments, provided are methods for the discovery and evolution of metabolic pathways with higher titers and/or lower toxicities, wherein starting pathways are mutated and/or reconfigured to create a library of pathways, and said library of pathways is screened using MADE to identify pathways that (i) produce higher quantities of inhibitor or activator than the starting pathway and/or (ii) exhibit a lower toxicity than the starting pathway ( FIG.  7   ). For example, mutagenized and/or reconfigured pathways may contain genes for a mutant enzyme, for example, a terpene synthase, that exhibits a higher activity than the wild-type enzyme; alternatively, mutagenized and/or reconfigured pathways may contain genes for a mutant terpene synthase that is more soluble or otherwise less toxic than a wild-type enzyme. 
     Some aspects of this disclosure provide molecules that inhibit protein tyrosine phosphatases (PTPs), for example, protein tyrosine phosphatase 1B (PTP1B;  FIGS.  9  and  10   ). Examples include amorphadiene and derivatives, taxadiene and derivatives, β-bisabolene and derivatives, α-bisabolene and derivatives, and α-longipinene and derivatives. In some embodiments, these molecules are provided as drugs or drug leads for the treatment of diseases to which PTPs contribute, for example, type 2 diabetes 42 , HER2-positive breast cancer 43 , or Rett syndrome 44 , as are methods of treatment of such diseases by administering an effective amount of the molecule(s) to a subject in need of such treatment. 
     Also provided are compositions or systems that include a population of host cells that comprise a protein of interest and a population of expression vectors comprising different metabolic pathways, wherein a cell or subset of the population of host cells produce a detectable output when the metabolic pathway produces a product that modulates the protein of interest, and optionally wherein the expression vectors yield detectable outputs higher than the output of a reference vector that harbors a reference pathway, for example, a vector that encodes a pathway that does not produce molecules with concentrations and/or potencies sufficient to modulate the activity of a protein of interest, in the cell or the subset of the population of host cells. 
     In some embodiments, the host cells comprise a genetically encoded system in which the activity of a protein of interest controls the assembly of a protein complex with an activity that is not possessed by either of two or more components of the complex and, thus, yields a detectable output in proportion to the amount of complex formed. In some embodiments, the protein of interest is an enzyme that adds a post-translational modification that causes two proteins, which are initially dissociated, to be covalently linked or to form a noncovalent complex. In some embodiments, the complex is formed by two proteins with a dissociation constant (K d ) less than or equal to the K d  of the complexes formed between SH2 domains and their phosphorylated substrates. 
     In some embodiments, the metabolic pathways encoded by the expression vectors produce phenylpropanoids or nonribosomal peptides. In some embodiments, the expression vectors comprising different metabolic pathways comprise a library of pathways generated by mutating one or more genes within a starting metabolic pathway. In some embodiments, one or more of the metabolic pathways comprises a set of genes of unknown biosynthetic capability. 
     In some embodiments, one or more of the metabolic pathways that produces a detectable output higher than the output of the reference pathway produces a product that differs from the products of other metabolic pathways. In some embodiments, one or more of the metabolic pathways that produces a detectable output higher than the output of the reference pathway produces a larger quantity of a product than the quantity of product generated by other metabolic pathways. In some embodiments, one or more of the metabolic pathways that produces a detectable output higher than the output of the reference pathway exhibits a lower cellular toxicity than other metabolic pathways. 
     In some embodiments, the protein of interest is a ubiquitin ligase, a SUMO transferase, a methyltransferase, a demethylase, an acetyltransferase, a glycosyltransferase, a palmitoyltransferase, or a related hydrolase. 
     Also provided herein are kits that include a population of expression vectors as described herein. In some embodiments, the kits also include the population of host cells that comprise a protein of interest as described herein. 
     The summary above is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology described herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the Detailed Description, Drawings, Examples, and Claims. 
     Definitions 
     The term “metabolic pathway,” as used herein, refers to a collection of genes that enable the synthesis of metabolite. 
     The term “metabolite,” as used herein, refers to an organic molecule assembled within a living system. 
     The term “small molecule,” as used herein, refers to a molecule with a molecular weight less than 900 daltons. 
     The term “phenylpropanoids,” as used herein, refers to an organic compound synthesized from the amino acids phenylalanine and/or tyrosine. 
     The term “nonribosomal peptide,” as used herein, refers to peptides synthesized without messenger RNA. For example, peptides synthesized from nonribosomal peptide synthases. 
     The term “modulator,” as used herein, refers to a molecule, peptide, protein, polynucleotide, or entity that changes the activity of another molecule, peptide, protein, polynucleotide, or entity. 
     The term “inhibitor,” as used herein, refers to a small molecule that reduces the activity of an enzyme. 
     The term “activator,” as used herein, refers to a small molecule that increases the activity of an enzyme. 
     The term “natural product,” as used herein, refers to a chemical compound or substance produced by a living organism. 
     The term “detection system,” as used herein, refers to a system that links the activity of a target enzyme to a detectable output. 
     The term “bacterial two-hybrid (B2H) system,” as used herein, refers to a genetically encoded system that links a protein-protein interaction to a detectable output. 
     The term “detectable output,” as used herein, refers to an output that can be detected with standard analytical instrumentation. Examples include fluorescence, luminescence, antibiotic resistance, or microbial growth. 
     The term “split protein,” as used herein, refers to a protein that exists as two separate halves, which, upon reassembly, restore the function of the protein. 
     The term “substrate domain,” as used herein, refers to a protein that includes a peptide fragment or protein component acted upon by a protein of interest. For example, a substrate domain may include the peptide fragment of a receptor protein targeted by a kinase or phosphatase of interest. 
     The term “vector,” as used herein, refers to a deoxyribonucleic acid (DNA) molecule used as a vehicle to artificially carry foreign genetic material into a cell. 
     The term “host cell,” as used herein, refers to a cell that can host the genetically encoded systems, on vectors or genomes, necessary for MADE. For example, as host cell may contain plasmids that encode both (i) a genetically encoded detection system that links the activity of a target enzyme to a detectable output and (ii) a metabolic pathway capable of synthesizing molecules that might or might not inhibit said target enzyme. 
     EXAMPLES 
     Example 1 
     In previous work, a strain of  E. coli  was generated with two genetically encoded modules—a B2H system that links the inhibition of PTP1B to the expression of a gene for antibiotic resistance, and a metabolic pathway for the production of amorphadiene—exhibited greater antibiotic resistance that similar strains with different metabolic pathways ( FIG.  2   ). In recent work, this result was explored further. First, it was shown that maximal resistance required both an active amorphadiene synthase (ADS) and a functional B2H system ( FIG.  9   ). Second, the inhibitory effect of amorphadiene, the dominant product of ADS, was confirmed by measuring its influence on PTP1B-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP;  FIG.  10 C ). Initial rates exhibited a saturation behavior characteristic of noncompetitive or uncompetitive inhibition; most importantly, the IC 50  for amorphadiene was ˜53 μM, a concentration lower than the 72 μM generated in liquid culture. For comparison, the IC 50  for taxadiene was 119 μM, a concentration far lower than its titer in liquid culture. Results of the in vitro studies thus indicate that amorphadiene confers antibiotic resistance by inhibiting PTP1B. Finally, an enzyme-linked immunosorbent assay (ELISA) was used to demonstrate the ability of amorphadiene to inhibit PTP1B inside of a HEK293T/17 cell ( FIG.  10 D- 10 E ). 
     The microbial system provides an interesting opportunity to explore how metabolic pathways evolve to generate functional molecules. To look for evolutionarily accessible changes in the activities ADS and GHS that improve their ability to generate inhibitors of PTP1B, mutants of both enzymes were prepared. For ADS, error-prone PCR and site-saturation mutagenesis of poorly conserved residues was used; for GHS, site-saturation mutagenesis of the wild-type enzyme was paired with a screen of several previously developed mutants with distinct product profiles 47  ( FIGS.  7 A,  7 B ). At least one mutant from each library consistently conferred survival at higher antibiotic concentrations than the wild-type enzyme ( FIG.  7 C,  7 D ). 
     The G34S/K51N mutant of ADS, which improved antibiotic resistance more than other mutants, is particularly intriguing because its mutated residues are located outside of the active site and alter neither product profile nor titer ( FIG.  7 E,  7 F ). It was hypothesized that these mutations might reduce a minor growth deficiency caused by heterologous ADS expression (e.g., they might reduce the formation of inclusion bodies). To test this hypothesis, the survival conferred by wild-type and mutant strains in the presence of an inactive B2H system was compared; the mutant strain showed more robust growth at high concentrations of antibiotic ( FIG.  7 G ). These results suggest that the engineered strain can select for less toxic enzyme mutants which, in the presence of other stresses, might improve production of inhibitory metabolites. 
     Intriguingly, the mutants of GHS that conferred enhanced antibiotic resistance (relative to the wild-type enzyme) altered product profile and/or titer ( FIGS.  7 H and  7 I ). Two examples include GHS A336C/T445C/S484C/I562L/M565L  (or ALP), which primarily generates α-longipinene, and GHS A319Q , which enhances terpenoid titer by ˜tenfold. The GHS mutants thus indicate that the engineered strain can select for enzyme mutants that generate different products and/or higher titers than a starting wild-type enzyme. 
     To expand the study, the survival conferred by terpene synthases that primarily generate β-bisabolene and α-bisabolene was also examined. Both of these enzymes enhanced antibiotic resistance; strikingly, kinetic studies of α-bisabolene purified from culture supernatant indicate that this molecule is particularly potent (i.e., IC 50 ˜20 μM in 10% DMSO;  FIG.  11   ). 
     The results of the analyses of terpene synthases suggest that amorphadiene and derivatives, taxadiene and derivatives, α-longipinene and derivatives, β-bisabolene and derivatives, and α-bisabolene and derivatives, and may provide an important source of pharmaceutically relevant PTP inhibitors. 
     Methods 
     Bacterial strains.  E. coli  DH10B, chemically competent NEB Turbo, or electrocompetent One Shot Top10 (Invitrogen) were used to carry out molecular cloning and to perform preliminary analyses of terpenoid production;  E. coli  BL2-DE31 were used to express proteins for in vitro studies; and  E. coli  s1030 48  were used for luminescence studies and for all experiments involving terpenoid-mediated growth (i.e., evolution studies). 
     For all strains, chemically competent cells were generated by carrying out the following steps: (i) each strain was plated on LB agar plates with the required antibiotics. (ii) One colony of each strain was used to inoculate 1 mL of LB media (25 g/L LB with appropriate antibiotics listed in TABLE 2) in a glass culture tube, and this culture was grew overnight (37° C., 225 RPM). (iii) The 1-mL culture was used to inoculate 100-300 mL of LB media (as above) in a glass shake flask, and this culture was grown for several hours (37° C., 225 RPM). (iv) When the culture reached an OD of 0.3-0.6, the cells were centrifuged (4,000×g for 10 minutes at 4° C.), the supernatant was removed, and the cells were resuspended in 30 mL of ice cold TFB1 buffer (30 mM potassium acetate, 10 mM CaCl 2 , 50 mM MnCl 2 , 100 mM RbCl, 15% v/v glycerol, water to 200 mL, pH=5.8, sterile filtered), and the suspension was incubated at 4° C. for 90 min. (v) Step iv was repeated, but resuspended in 4 mL of ice cold TFB2 buffer (10 mM MOPS, 75 mM CaCl 2 , 10 mM RbCl 2 , 15% glycerol, water to 50 mL, pH=6.5, sterile filtered). (iv) The final suspension as split into 100 aliquots and frozen at −80° C. until further use. 
     Electrocompetent cells were generated by following an approach similar to the one above. In step iv, however, the cells were resuspended in 50 mL of ice cold MilliQ water and repeated this step twice—first with 50 mL of 20% sterile glycerol (ice cold) and, then, with 1 mL of 20% sterile glycerol (ice cold). The pellets were frozen as before. 
     Materials. Methyl abietate was purchased from Santa Cruz Biotechnology; trans-caryophyllene, farnesol, tris(2-carboxyethyl)phosphine (TCEP), bovine serum albumin (BSA), M9 minimal salts, phenylmethylsulfonyl fluoride (PMSF), and DMSO (dimethyl sulfoxide) were purchased from Millipore Sigma; glycerol, bacterial protein extraction reagent II (B-PERII), and lysozyme from were purchased VWR; cloning reagents were purchased from New England Biolabs; amorphadiene was purchased from Ambeed, Inc.; and all other reagents (e.g., antibiotics and media components) were purchased from Thermo Fisher. Taxadiene was a kind gift from Phil Baran of the The Scripps Research Institute. Mevalonate was prepared by mixing 1 volume of 2 M DL-mevalanolactone with 1.05 volumes of 2 M KOH and incubating this mixture at 37° C. for 30 minutes.
 
Cloning and molecular biology. All plasmids were constructed by using standard methods (i.e., restriction digest and ligation, Golden Gate and Gibson assembly, Quikchange mutagenesis, and circular polymerase extension cloning). TABLE 1 describes the source of each gene; TABLES 2 and 3 describe the composition of all final plasmids.
 
     Construction of the B2H system was begun by integrating the gene for HA4-rpoZ from pAB094a into pAB078d and by replacing the ampicillin resistance marker of pAB078d with a kanamycin resistance marker (Gibson Assembly). The resulting “combined” plasmid was modified, in turn, by replacing the HA4 and SH2 domains with kinase substrate and substrate recognition (i.e., SH2) domains, respectively (Gibson assembly), and by integrating genes for Src kinase, CDC37, and PTP1B in various combinations (Gibson assembly). The functional B2H system was finalized by modifying the SH2 domain with several mutations known to enhance its affinity for phosphopeptides (K15L, T8V, and C10A, numbered as in Kaneko et. al. 40 ), by exchanging the GOI for luminescence (LuxAB) with one for spectinomycin resistance (SpecR), and by toggling promoters and ribosome binding sites to enhance the transcriptional response (Gibson assembly and Quickchange Mutagenesis, Agilent Inc.). Note: For the last step, Prol to ProD was also converted by using the Quikchange protocol. When necessary, plasmids with arabinose-inducible components were constructed by cloning a single component from the B2H system into pBAD (Golden Gate assembly). TABLES 4 and 5 list the primers and DNA fragments used to construct each plasmid. 
     Pathways for terpenoid biosynthesis were assembled by purchasing plasmids encoding the first module (pMBIS) and sesquiterpene synthases (ADS or GHS in pTRC99a) from Addgene, and by building the remaining plasmids. Genes for ABS, TXS, and GGPPS were integrated into pTRC99t (i.e., pTRC99a without BsaI sites), and a version of pADS was modified by adding a gene for P450 BM3  with three mutations that enable the epoxidation of amorphadiene (F87A, R47L, and Y51F; P450G3; Gibson Assembly and Quickchange Mutagenesis) 49 . TABLE 6 lists the primers and DNA fragments used to construct each plasmid. 
     Luminescence assays. Preliminary B2H systems (which contained LuxAB as the GOI) were characterized with luminescence assays. In brief, necessary plasmids were transformed into  E. coli  s1030 (TABLE 2), the transformed cells were plated onto LB agar plates (20 g/L agar, 10 g/L tryptone, 10 g/L sodium chloride, and 5 g/L yeast extract with antibiotics described in TABLE 2), and all plates were incubated overnight at 37° C. Individual colonies were used to inoculate 1 ml of terrific both (TB at 2%, or 12 g/L tryptone, 24 g/L yeast extract, 12 mL/L 100% glycerol, 2.28 g/L KH 2 PO 4 , 12.53 g/L K 2 HPO 4 , pH=7.0, and antibiotics described in TABLE 2), and we incubated these cultures overnight (37° C. and 225 RPM). The following morning, each culture was diluted by 100-fold into 1 ml of TB media (above), and these cultures were incubated in individual wells of a deep 96-well plate for 5.5 hours (37° C., 225 RPM). (Note: When pBAD was present, the TB media was supplemented with 0-0.02 w/v % arabinose). An amount of 100 μL of each culture was transferred into a single well of a standard 96-well plate and measured both OD 600  and luminescence (gain: 135, integration time: 1 second, read height: 1 mm) on a Biotek Synergy plate reader. Analogous measurements of cell-free media were performed to measure background signals, which were subtracted from each measurement prior to calculating OD-normalized luminescence (i.e., Lum/OD 600 ).
 
Analysis of antibiotic resistance. The spectinomycin resistance conferred by various B2H systems in the absence of terpenoid pathways was evaluated by carrying out the following steps: (i)  E. coli  were transformed with the necessary plasmids (TABLE 2) and the transformed cells were plated onto LB agar plates (20 g/L agar, 10 g/L tryptone, 10 g/L sodium chloride, 5 g/L yeast extract, 50 μg/ml kanamycin, 10 μg/ml tetracycline). (ii) Individual colonies were used to inoculate 1-2 ml of TB media (12 g/L tryptone, 24 g/L yeast extract, 12 mL/L 100% glycerol, 2.28 g/L KH 2 PO 4 , 12.53 g/L K 2 HPO 4 , 50 μg/ml kanamycin, 10 μg/ml tetracycline, pH=7.0), and these cultures were incubated overnight (37° C., 225 RPM). In the morning, each culture was diluted by 100-fold into 4 ml of TB media (as above) with 0-500 μg/ml spectinomycin (spectinomycin was used only for the results depicted in  FIG.  14   ), and these cultures were incubated in deep 24-well plates until wells containing 0 μg/ml spectinomycin reached an OD 600  of 0.9-1.1. (iv) Each 4-ml culture was diluted by 10-fold into TB media with no antibiotics and plated 10-μL drops of the diluent onto agar plates with various concentrations of spectinomycin. (v) Plates were incubated overnight (37° C.) and photographed the following day.
 
     To examine terpenoid-mediated resistance, steps i and ii were performed as described above with the addition of 34 μg/ml chloramphenicol and 50 μg/ml carbenicillin in all liquid/solid media. The experiment then proceeded with the following steps: (iii) Samples were diluted from 1-ml cultures to an OD 600  of 0.05 in 4.5 ml of TB media (supplemented with 12 g/L tryptone, 24 g/L yeast extract, 12 mL/L 100% glycerol, 2.28 g/L KH 2 PO 4 , 12.53 g/L K 2 HPO 4 , 50 μg/ml kanamycin, 10 μg/ml tetracycline, 34 μg/ml chloramphenicol, and 50 μg/ml carbenicillin), which were incubated in deep 24-well plates (37° C., 225 RPM). (iv) At an OD 600  of 0.3-0.6, 4 ml of each culture was transferred to a new well of a deep 24-well plate, 500 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 20 mM of mevalonate was added, and incubated for 20 hours (22° C., 225 RPM). (v) Each 4-ml culture was diluted to an OD 600  of 0.1 with TB media and plated 10 μL of the diluent onto either LB or TB plates supplemented with 500 μM IPTG, 20 mM mevalonate, 50 μg/ml kanamycin, 10 μg/ml tetracycline, 34 μg/ml chloramphenicol, 50 μg/ml carbenicillin, and 0-1200 μg/ml spectinomycin (for both plates, 20 g/L agar was used with media and buffer components described above). Note: to control the range of antibiotic resistance, LB plates were used for ADS and its mutants, and TB plates, which improve terpenoid titers, were used for GHS and its mutants. (iv) All plates were incubated at 30° C. and photographed after 2 days. 
     Terpenoid biosynthesis.  E. coli  were prepared for terpenoid production by transforming cells with plasmids harboring requisite pathway components (TABLE 2) and plating them onto LB agar plates (20 g/L agar, 10 g/L tryptone, 10 g/L sodium chloride, and 5 g/L yeast extract with antibiotics described in TABLE 2). One colony from each strain was used to inoculate 2 ml TB (12 g/L tryptone, 24 g/L yeast extract, 12 mL/L 100% glycerol, 2.28 g/L KH 2 PO 4 , 12.53 g/L K 2 HPO 4 , pH=7.0, and antibiotics described in TABLE 2) in a glass culture tube for ˜16 hours (37° C. and 225 RPM). These cultures were diluted by 75-fold into 10 ml of TB media and the new cultures were incubated in 125 mL glass shake flasks (37° C. and 225 RPM). At an OD 600  of 0.3-0.6, 500 μM IPTG and 20 mM mevalonate were added. After 72-88 hours of growth (22° C. and 225 RPM), terpenoids were extracted from each culture. 
     To measure terpenoid production over time, the approach described above was used with the following modifications: (i) Overnight cultures were diluted with 1:75 mL in 4.5 mL TB supplemented with antibiotics in a glass culture tube. (ii) When cultures reached an OD 600  of 0.3-0.6, 4 mL of each culture were moved to a new culture tube and 500 μM IPTG, 20 mM mevalonate, 0-800 μg/mL spectinomycin, and 1 mL dodecane were added (to extract terpenoids). Every 4 hours, 100 μL of the dodecane sample was removed for GC/MS analysis. 
     Protein expression and purification. PTPs were expressed and purified as described previously 42 . Briefly,  E. coli  BL21(DE3) cells were transformed with pET21b vectors, and induced with 500 μM IPTG at 22° C. for 20 hours. PTPs were purified from cell lysate by using desalting, nickel affinity, and anion exchange chromatography (HiPrep 26/10, HisTrap HP, and HiPrep Q HP, respectively; GE Healthcare). The final protein (30-50 μM) was stored in HEPES buffer (50 mM, pH 7.5, 0.5 mM TCEP) in 20% glycerol at −80° C.
 
Extraction and purification of terpenoids. Hexane was used to extract terpenoids generated in liquid culture. For 10-mL cultures, 14 mL of hexane was added to 10 ml of culture broth in 125-mL glass shake flasks, the mixture (100 RPM) shaken for 30 minutes, centrifuged (4000×g), and 10 mL of the hexane layer was withdrawn for further analysis. For 4-mL cultures, 600 μL hexane were added to 1 mL of culture broth in a microcentrifuge tube, the tubes were vortexed for 3 minutes, the tubes were centrifuged for 1 minute (17000×g), and 300-400 μL of the hexane layer was saved for further analysis.
 
     To purify amorphadiene, 500-1000 mL culture broth was supplemented with hexane (16.7% v/v), the mixture was shaken for 30 minutes (100 RPM), the hexane layer was isolated with a separatory funnel, the isolated organic phase was centrifuged (4000×g), and the hexane layer withdrawn. To concentrate the terpenoid products, excess hexane was evaporated in a rotary evaporator to bring the final volume to 500 μL, and the resulting mixture was passed over a silica gel one or two times (Sigma-Aldrich; high purity grade, 60 Å pore size, 230-400 mesh particle size)). Elution fractions (100% hexane) were analyzed on the GC/MS and pooled fractions with the compound of interest (amorphadiene). Once purified, pooled fractions were dried under a gentle stream of air, the terpenoid solids were resuspended in DMSO, and the final samples were quantified as outlined below. 
     GC-MS analysis of terpenoids. Terpenoids generated in liquid culture were measured with a gas chromatograph/mass spectrometer (GC-MS; a Trace 1310 GC fitted with a TG5-SilMS column and an ISQ 7000 MS; Thermo Fisher Scientific). All samples were prepared in hexane (directly or through a 1:100 dilution of DMSO) with 20 μg/ml of caryophyllene or methyl abietate as an internal standard. When the peak area of an internal standard exceeded ±30% of the average area in hexane samples containing only standard, the corresponding samples were re-analyzed. For all runs, the following GC method was used: hold at 80° C. (3 min), increase to 250° C. (15° C./min), hold at 250° C. (6 min), increase to 280° C. (30° C./min), and hold at 280° C. (3 min). To identify various analytes, m/z ratios were scanned from 50 to 550. 
     Sesquiterpenes generated by variants of ADS were examined by using select ion mode (SIM) to scan for the molecular ion (m/z=204). For quantification, we used Eq. 1: 
     
       
         
           
             
               
                 
                   
                     C 
                     i 
                   
                   = 
                   
                     
                       C 
                       std 
                     
                     * 
                     
                       
                         A 
                         i 
                       
                       
                         A 
                         std 
                       
                     
                     * 
                     R 
                   
                 
               
               
                 
                   ( 
                   
                     Eq 
                     . 
                         
                     1 
                   
                   ) 
                 
               
             
           
         
       
       
         
           
             
               
                 
                   R 
                   = 
                   
                     
                       
                         A 
                         
                           std 
                           , 
                           o 
                         
                       
                       / 
                       
                         C 
                         
                           std 
                           , 
                           o 
                         
                       
                     
                     
                       
                         A 
                         
                           ref 
                           , 
                           o 
                         
                       
                       / 
                       
                         C 
                         
                           ref 
                           , 
                           o 
                         
                       
                     
                   
                 
               
               
                 
                   ( 
                   
                     Eq 
                     . 
                         
                     2 
                   
                   ) 
                 
               
             
           
         
       
     
     where A i  is the area of the peak produced by analyte i, A std  is the area of the peak produced by C std  of caryophyllene in the sample, and R is the ratio of response factors for caryophyllene and amorphadiene in a reference sample. 
     Sesquiterpenes generated by variants of GHS were quantified by using the aforementioned procedure with several modifications: Methyl abietate was used as an internal standard (several mutants of GHS generate caryophyllene as a product); both m/z=204 and m/z=121, a common ion between sesquiterpenes and methyl abietate were scanned for; a ratio of response factors for amorphadiene and methyl abietate at m/z=121 for R was used; and peak areas were calculated at m/z=121. For all analyses, the analysis was focused on peaks with areas that exceeded 1% of the total area of all peaks at m/z=204. 
     Diterpenoids were quantified by, once again, accompanying the general procedure with several modifications: A different molecular ion (m/z=272) and an ion common to both diterpenoids and caryophyllene (m/z=93) was scanned for; a ratio of response factors for pure taxadiene (a kind gift from Phil Baran) and caryophyllene at m/z=93 was used; and peak areas m/z=93 were calculated. For all analyses, only peaks with areas that exceeded 1% of the total area of all peaks at m/z=272 were examined. 
     Molecules were identified by using the NIST MS library and, when necessary, this identification was confirmed with analytical standards or mass spectra reported in the literature. Note: The assumption of a constant response factor for different terpenoids (e.g., all sesquiterpenes and diterpenes ionize like amorphadiene and taxadiene, respectively) can certainly yield error in estimates of their concentrations; the analyses described herein, which are consistent with those of other studies of terpenoid production in microbial systems 50,51 , thus supply rough estimates of concentrations for all compounds except amorphadiene and taxadiene (which had analytical standards). 
     Homology modeling of ADS and GHS. Homology models of ADS and GHS were constructed by using SWISS-MODEL with structures for α-bisabolol synthase (pdb entry 4gax) and α-bisabolene synthase (pdb entry 3sae) as templates, respectively 52 . This software package uses ProMod3 to build models from a target-template alignment, which preserves the structures of conserved regions and remodels insertions and deletions with a fragment library 53,54 .
 
Preparation of mutant libraries. Libraries of enzyme mutants were prepared by using site-saturation mutagenesis (SSM) and error-prone PCR (ePCR). For SSM, the following steps were performed: (i) Genes were amplified with NNK primers that targeted select sites. (ii) The amplified genes were digested with DpnI, purified with gel electrophoresis, and either Gibson Assembly or circular polymerase extension cloning (CPEC) 55  was used to integrate them into plasmids (pTS xx ). (iii) Heat shock was used to transform the fully assembled plasmids into chemically competent NEB Turbo cells. (iv) Library size was determined by plating dilutions of the transformation reactions on several LB agar plates (20 g/L agar, 10 g/L tryptone, 10 g/L sodium chloride, 5 g/L yeast extract, 50 μg/ml carbenicillin), and all remaining cells were plated over 9-10 plates for subsequent analysis. (v) Colonies were sequenced to verify that at least 5 of 6 transformants contained mutated genes. (vi) Plates were scraped into LB media (25 g/L LB broth mix, no antibiotics) and the final transformants were miniprepped to recover the DNA Library. (vii) All final libraries were frozen in MilliQ water at −20° C.
 
     For ePCR, the Genemorph II kit (Agilent) was used with ˜0.5-2.5 mutations/kb. The final plasmids were dialyzed and electroporated into One Shot electrocompetent Top 10 cells, and the final plasmids were sequenced, extracted, and stored as described above. 
     Analysis of mutant libraries. Each mutant library was screened by carrying out the following steps: (i) 100 ng of each site-specific SSM library for a given terpene synthase was pooled. (ii) Each complete library (i.e., ePCR or pooled SSM) was dialyzed for 2 hours. (iii) Up to 10 μL (&lt;1 μg) of each library was electroporated into a strain of  E. coli  harboring both the pMBIS pathway and the B2H system. (iv) 1 mL of SOC was added to the transformed cells and incubated for 1 hour (37° C. and 225 RPM). (v) 100 μL of the SOC outgrowth was serial diluted and plated onto LB agar plates (20 g/L agar, 10 g/L tryptone, 10 g/L sodium chloride, 5 g/L yeast extract, 50 μg/ml carbenicillin, 10 μg/ml tetracycline, 50 μg/ml kanamycin, and 34 μg/ml chloramphenicol) and the plates were incubated overnight (37° C.). This step allowed for quantification of the number of transformants screened (i.e., a number determined by counting colonies). (vi) The remaining 900 μL of transformed cells was added to 100 mL of TB (12 g/L tryptone, 24 g/L yeast extract, 12 mL/L 100% glycerol, 2.28 g/L KH 2 PO 4 , 12.53 g/L K 2 HPO 4 , 50 μg/ml carbenicillin, 10 μg/ml tetracycline, 34 μg/ml chloramphenicol, 50 μg/ml kanamaycin, pH=7.0) in 500-mL Erlenmeyer flasks, and these flasks were incubated overnight (37° C. and 225 RPM). (vii) In the morning, an aliquot of each culture was diluted to an OD 600  of 0.05 in 4 mL of TB and incubated in glass culture tubes (37° C. and 225 RPM). (viii) At an OD 600  of 0.3-0.6, terpenoid production was induced by adding 5-20 mM mevalonate and 500 μM IPTG, and the resulting cultures were incubated for 20 hours (22° C. and 225 RPM). (ix) Each culture was diluted to an OD 600  of 0.001 and 100 μL of diluent was plated onto agar plates containing 500 μM IPTG, 5-20 mM mevalonate, 50 μg/ml kanamycin, 10 μg/ml tetracycline, 34 μg/ml chloramphenicol, 50 μg/ml carbenicillin, and 0-1000 μg/ml spectinomycin. (x) Colonies that survived high concentrations of spectinomycin were used to inoculate 4 mL of LB media (25 g/L LB broth mix, 50 μg/ml carbenicillin, 10 μg/ml tetracycline, 34 μg/ml chloramphenicol, 50 μg/ml kanamaycin, which was incubated overnight (37° C., 225 RPM). (xi) Plasmid DNA was extracted from the overnight culture for Sanger sequencing. 
     The influence of interesting mutations—and a check for false positive—were confirmed by rescreening them in freshly prepared mutants. Site directed mutagenesis was used to introduce mutations found in the hits and then their antibiotic resistance was analyzed using the drop-based plating method described above. 
     Enzyme kinetics. To examine terpenoid-mediated inhibition, PTP1B-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) was measured in the presence of various concentrations of terpenoids. Each reaction included PTP1B (0.05 μM), pNPP (0.33, 0.67, 2, 5, 10, and 15 mM), inhibitors (110 μM, 50 μM, and 15 μM for amorphadiene; 100 μM, 50 μM, and 16.7 μM for taxadiene), and buffer (50 mM HEPES pH=7.5, 0.5 mM TCEP, 50 μg/ml BSA, 10% DMSO). The formation of p-nitrophenol was monitored by measuring absorbance at 405 nm every 10 seconds for 5 minutes on a Spectramax M2 plate reader. 
     Kinetic models were evaluated in three steps: (i) Initial-rate measurements collected in the absence and presence of inhibitors were fitted to Michaelis-Menten and inhibition models, respectively (here, the nlinfit and fminsearch functions from MATLAB were used). (ii) An F-test was used to compare the mixed model to the single-parameter model with the least sum squared error (here, the fcdf function from MATLAB was used to assign p-values), and the mixed model was accepted when p&lt;0.05. (iii) The Akaike&#39;s Information Criterion (AIC) was used to compare the best-fit single parameter model to each alternative single parameter model, and the “best-fit” model was accepted when the difference in AIC (Δ i ) exceed 10 for all comparisons. 56  Note: For amorphadiene, this criterion was not met; both noncompetitive and uncompetitive models, however, yielded indistinguishable IC 50 &#39;s. 
     The half maximal inhibitory concentration (IC 50 ) of inhibitors were estimated by using the best-fit kinetic models to determine the concentration of inhibitor required to reduce initial rates of PTP-catalyzed hydrolysis of 15 mM of pNPP by 50%. The MATLAB function “nlparci” was used to determine the confidence intervals of kinetic parameters, and those intervals were propagated to estimate corresponding confidence on IC 50 &#39;s. 
     REFERENCES FOR EXAMPLE 1 
     
         
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     Example 2 
     The design of small molecules that inhibit disease-relevant proteins represents a longstanding challenge of medicinal chemistry. Here, we describe an approach for encoding this challenge—the inhibition of a human drug target—into a microbial host and using it to guide the discovery and biosynthesis of targeted, biologically active natural products. This approach identified two previously unknown terpenoid inhibitors of protein tyrosine phosphatase 1B (PTP1B), an elusive therapeutic target for the treatment of diabetes and cancer. At least one inhibitor targets an allosteric site, which confers unusual selectivity; both can inhibit PTP1B in living cells. A screen of 24 uncharacterized terpene synthases from a pool of 4,464 genes uncovered additional hits, demonstrating a scalable discovery approach, and the incorporation of different PTPs into the microbial host yielded PTP-specific detection systems. Findings illustrate the potential for using microbes to discover and build natural products that exhibit precisely defined biochemical activities yet possess unanticipated structures and/or binding sites. 
     Despite advances in structural biology and computational chemistry, the design of small molecules that bind tightly and selectively to disease-relevant proteins remains exceptionally difficult 1 . The free energetic contributions of rearrangements in the molecules of water that solvate binding partners and structural changes in the binding partners themselves are particularly challenging to predict and, thus, to incorporate into molecular design 2,3 . Drug development, as a result, often begins with screens of large compound libraries 4 . 
       Nature  has endowed living systems with the catalytic machinery to build an enormous variety of biologically active molecules—a diverse natural library 5 . These molecules evolved to carry out important metabolic and ecological functions (e.g., the phytochemical recruitment of predators of herbivorous insects 6 ) but often also exhibit useful medicinal properties. Over the years, screens of environmental extracts and natural product libraries—augmented, on occasion, with combinatorial (bio)chemistry 7-9 —have uncovered a diverse set of therapeutics, from aspirin to paclitaxel 10 . Unfortunately, these screens tend to be resource intensive 11 , limited by low natural titers 12 , and largely subject to serendipity 13 . Bioinformatic tools, in turn, have permitted the identification of biosynthetic gene clusters 14,15 , where co-localized resistance genes can reveal the biochemical function of their products 16,17 . The therapeutic applications of many natural products, however, differ from their native functions 18 , and many biosynthetic pathways can, when appropriately reconfigured, produce entirely new and, perhaps, more effective therapeutic molecules 19,20 . Methods for efficiently identifying and building natural products that inhibit specific disease-relevant proteins remain largely undeveloped. 
     Protein tyrosine phosphatases (PTPs) are an important class of drug targets that could benefit from new approaches to inhibitor discovery. These enzymes catalyze the hydrolytic dephosphorylation of tyrosine residues and, together with protein tyrosine kinases (PTKs), contribute to an enormous number of diseases (e.g., cancer, autoimmune disorders, and heart disease, to name a few) 21,22 . The last several decades have witnessed the construction of many potent inhibitors of PTKs, which are targets for over 30 approved drugs 23 . Therapeutic inhibitors of PTPs, by contrast, have proven difficult to develop. These enzymes possess well conserved, positively charged active sites that make them difficult to inhibit with selective, membrane-permeable molecules 24 ; they lack targeted therapeutics of any kind. 
     In this study, we describe an approach for using microbial systems to find natural products that inhibit difficult-to-drug proteins. We focused on protein tyrosine phosphatase 1B (PTP1B), a therapeutic target for the treatment of type 2 diabetes, obesity, and HER2-positive breast cancer 25 . PTP1B possesses structural characteristics that are generally representative of the PTP family 26  and regulates a diverse set of physiological processes (e.g., energy expenditure 27 , inflammation 28 , and neural specification in embryonic stem cells 29 ). In brief, we assembled a strain of  Escherichia coli  with two genetic modules—(i) one that links cell survival to the inhibition of PTP1B and (ii) one that enables the biosynthesis of structurally varied terpenoids. In a study of five well-characterized terpene synthases, this strain identified two previously unknown terpenoid inhibitors of PTP1B. Both inhibitors were selective for PTP1B, exhibited distinct binding mechanisms, and increased insulin receptor phosphorylation in mammalian cells. A screen of 24 uncharacterized terpene synthases from eight phylogenetically diverse clades uncovered additional hits, demonstrating a scalable approach for finding inhibitor-synthesizing genes. A simple exchange of PTP genes, in turn, permitted the facile extension of our genetically encoded detection system to new targets. Our findings illustrate a versatile approach for using microbial systems to find targeted, readily synthesizable inhibitors of disease-relevant enzymes. 
     Development of a Genetically Encoded Objective 
       E. coli  is a versatile platform for building natural products from unculturable or low-yielding organisms 30,31 . We hypothesized that a strain of  E. coli  programmed to detect the inactivation of PTP1B (i.e., a genetically encoded objective) might enable the discovery of natural products that inhibit it (i.e., molecular solutions to the objective). To program such a strain, we assembled a bacterial two-hybrid (B2H) system in which PTP1B and Src kinase control gene expression ( FIG.  21 A ). In this system, Src phosphorylates a substrate domain, enabling a protein-protein interaction that activates transcription of a gene of interest (GOI). PTP1B dephosphorylates the substrate domain, preventing that interaction, and the inactivation of PTP1B re-enables it.  E. coli  is a particularly good host for this detection system because its proteome is sufficiently orthogonal to the proteome of  H. sapiens  to minimize off-target growth defects that can result from the regulatory activities of Src and PTP1B (Note 1) 32 . 
     We carried out B2H development in several steps. To begin, we assembled a luminescent “base” system in which Src modulates the binding of a substrate domain to an Src homology 2 (SH2) domain ( FIG.  21 B ); this system, which includes a chaperone that helps Src to fold (Cdc37) 33 , is similar to other B2H designs that detect protein-protein binding 34 . Unfortunately, our initial system did not yield a phosphorylation-dependent transcriptional response, so we complemented it with inducible plasmids—each harboring a different system component—to identify proteins with suboptimal expression levels ( FIG.  21   b   ). Interestingly, secondary induction of Src increased luminescence, an indication that insufficient substrate phosphorylation and/or weak substrate-SH2 binding depressed GOI expression in our base system. We modified this system by swapping in different substrate domains, by adding mutations to the SH2 domain that enhance its affinity for phosphopeptides 35 , and by removing the gene for Src—a modification that allowed us to control expression exclusively from a second plasmid. With this configuration, induction of Src increased luminescence most prominently for the MidT substrate ( FIG.  1 C ), and simultaneous induction of both Src and PTP1B prevented that increase—an indication of intracellular PTP1B activity ( FIG.  21 D ). We finalized the MidT system by incorporating genes for PTP1B and Src, by adjusting promoters and ribosome binding sites to amplify its transcriptional response further ( FIG.  21 D ,  FIG.  13   , and  FIG.  14   ), and by adding a gene for spectinomycin resistance (SpecR) as the GOI. The final plasmid-borne detection system required the inactivation of PTP1B to permit growth at high concentrations of antibiotic ( FIG.  21 E ). 
     Biosynthesis of PTP1B Inhibitors 
     To search for inhibitors of PTP1B that bind outside of its active site, we coupled the B2H system with metabolic pathways for terpenoids, a structurally diverse class of secondary metabolites with largely nonpolar structures ( FIG.  22 A ), some of which are known to inhibit PTP1B 36,37 . Terpenoids include over 80,000 known compounds and represent nearly one-third of all characterized natural products 38  (the basis of approximately 50% of clinically approved drugs 39 ). To begin, we focused on a handful of structurally diverse terpenoids without established inhibitory effects ( FIG.  22 B ): Amorphadiene (AD),  -humulene, α-bisabolene (AB), abietadiene, and taxadiene. Each terpenoid pathway consisted of two plasmid-borne modules: (i) the mevalonate-dependent isoprenoid pathway from  S. cerevisiae  (optimized for expression in  E. coli   40 ) and (ii) a terpene synthase previously demonstrated to express and produce one of the five selected terpenoids in  E. coli   40-41 . The terpene synthase was supplemented, when necessary for diterpenoid production, with a geranylgeranyl diphosphate synthase. These modules generated terpenoids at titers of 0.3-18 mg/L in  E. coli  ( FIG.  26   ). 
     We screened each pathway for its ability to produce inhibitors of PTP1B by transforming  E. coli  with plasmids harboring both the pathway of interest and the B2H system ( FIG.  22 C ). To our surprise, pathways for AD and AB permitted survival at high concentrations of antibiotic. Critically, GC-MS traces confirmed that all pathways generated terpenoids in the presence of the B2H system ( FIG.  22 D ,  FIG.  26   ), and maximal resistance of the AD- and AB-producing strains required both an active terpene synthase and a functional B2H system ( FIG.  26 D ). 
     We confirmed the inhibitory effects of purified terpenoids by examining their influence on PTP1B-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP;  FIG.  22 E , TABLE 12). The IC 50 s for AD and AB were 53±8 μM and 13±2 μM, respectively, in 10% DMSO ( FIG.  22 F ). These IC 50 s are surprisingly strong for small, unfunctionalized hydrocarbons; the ligand efficiencies of both inhibitors are high (TABLE 15), and their potencies are similar to those of larger molecules that form hydrogen bonds and other stabilizing interactions with PTP1B 21,45 . Both IC 50 s are also similar to the respective terpenoid concentrations in liquid culture ( FIG.  22 G ), a finding consistent with in vivo inhibition (terpenoids tend to accumulate intracellularly 46 , so in vivo concentrations may be even higher). Our growth-coupled assays, kinetic assays, and production measurements, taken together, indicate that AD and AB activate the B2H system by inhibiting PTP1B inside the cell. 
     Biophysical Analysis of PTP1B Inhibitors 
     Allosteric inhibitors of PTPs are valuable starting points for drug development. These molecules bind outside of the well conserved, positively charged active sites of PTPs and tend to have improved selectivities and membrane permeabilities over substrate analogs 21 . Motivated by these considerations, an early screen identified a benzbromarone derivative that inhibited PTP1B weakly (IC 50 =350 μM) without competing with substrates; subsequent optimization of this compound led to two improved inhibitors (IC 50 &#39;s=8 and 22 μM) that bind to an allosteric site 45  ( FIG.  23 A ). Over the next 15 years, efforts to find new inhibitors that bind to this or other allosteric regions on the catalytic domain have been largely unsuccessful 47 . Benzbromarone derivatives are the only allosteric inhibitors with crystallographically verified binding sites. (Although, an allosteric inhibitor that binds to a disordered region of the full-length protein has been characterized with NMR 25 ). New approaches for finding allosteric inhibitors are clearly needed. 
     Our microbial system could grant access to new compounds that bind in unexpected ways. AD and AB provide examples. They are highly nonpolar and, thus, incapable of engaging in the hydrogen bonds and electrostatic interactions on which most other PTP inhibitors rely 21,45 . To examine their binding mechanisms in detail, we sought to collect X-ray crystal structures of PTP1B bound to AD and α-bisabolol, a soluble analogue of AB (a ligand for which poor solubility precluded soaking experiments). Unfortunately, only the structure of PTP1B bound to AD was sufficient for unambiguous determination of a binding site ( FIG.  30    and  FIG.  31   ). This inhibitor binds to the same allosteric site targeted by benzbromarone derivatives. Its binding mode, however, is distinct: (i) AD causes the α7 helix of PTP1B to reorganize to create a hydrophobic cleft ( FIG.  23 B ); this type of reorganization is interesting because it is typically slow (micro- to millisecond) 48  and difficult to incorporate into computational ligand design 49 . (ii) It likely adopts multiple bound conformations (i.e., the electron density indicates regions of disorder;  FIG.  30   ). This behavior, which is supported by molecular dynamics simulations, is consistent with prior work on the binding of proteins to hydrocarbon moieties, which tend to be “mobile” in their binding pockets. 
     We probed the binding of AD and AB further with several additional analyses. First, we examined the inhibition of PTP1B by dihydroartemisinic acid. This structural analogue of AD has a carboxyl group that, according to our crystal structure, should interfere with binding to the hydrophobic cleft created by the α7 helix ( FIG.  23 C ). The IC 50  of this molecule was eight-fold higher than that of AD, a reduction in potency consistent with its crystallographic pose ( FIG.  23   d    and  FIG.  33   ). Second, we studied the competition between AD and two inhibitors that bind to the active site: (i) TCS401, which causes the WPD loop to adopt a closed conformation, and (ii) orthovanadate, which does not. For background, benzobromarones, upon binding to the C-terminal allosteric site, stabilize the WPD loop in an open conformation that is incompatible with the binding of TCS401, but not orthovanadate. Our kinetic data suggest that AD behaves similarly ( FIG.  23 E  and  FIG.  23 F ), a finding consistent with a shared binding site and mechanism of modulation. Finally, we assessed the inhibitory effects of AD and AB against TC-PTP, the closest homolog of PTP1B. Intriguingly, both molecules inhibited TC-PTP five- to six-fold less potently than PTP1B ( FIG.  23 G  and  FIG.  33   ). This finding is consistent with binding to the poorly conserved allosteric site. Importantly, this selectivity may seem modest, but it matches or exceeds the selectivities of most pre-optimized inhibitors (including benzobromarone derivatives) and is exceedingly rare for unfunctionalized hydrocarbons 50 . We assessed the contribution of the α7 helix to selectivity, in turn, by removing the equivalent region from PTP1B and TC-PTP ( FIG.  23 G ). This modification caused a four-fold reduction in the selectivity of AD, an effect consistent with the involvement of the α7 helix in its binding. Intriguingly, the selectivity of AB was insensitive to this modification; the unambiguous determination of the binding site of this ligand requires additional data. 
     AD and AB are lipophilic molecules that could be valuable for their ability to pass through the membranes of mammalian cells. To examine the biological activity of these molecules, we incubated them with HEK293T/17 cells and used an enzyme-linked immunosorbent assay to measure shifts in insulin receptor (IR) phosphorylation. IR is a receptor tyrosine kinase that undergoes PTP1B-mediated dephosphorylation from the cytosolic side of the plasma membrane (PTP1B, in turn, localizes to the endoplasmic reticulum of the cell). Both molecules increased IR phosphorylation over a negative control ( FIG.  23 H  and  FIG.  35   ). We checked for off-target contributions to this signal, in turn, by repeating the ELISA with equivalent concentrations of dihydroartemisinic acid and α-bisabolol. To our satisfaction, both molecules led to a reduction in signal consistent with their reduced potencies. 
     Other PTPs can promote IR dephosphorylation; SHP1 and SHP2 provide two examples 51-53 . To examine the potential contribution of these enzymes to the increase in IR phosphorylation observed in our ELISA, we measured their inhibition by AD and AB. Briefly, AD inhibited SHP2 three-fold less potently than PTP1B, and its inhibition of SHP1 was too weak to measure ( FIGS.  34 A- 34 B ). The low potency of AB against SHP1 and SHP2 also precluded experimental measurement ( FIGS.  34 C- 34 D ). These potencies, together with the aforementioned analysis of weakly inhibitory structural analogs, suggest that the inhibition of PTP1B by AD and AB is the primary cause of the increase in IR phosphorylation observed in our ELISA experiments. 
     A Scalable Approach to Molecular Discovery 
     Our microbial strain provides a powerful tool for screening genes for their ability to generate novel PTP1B inhibitors. Most terpenoids, as a case study, are not commercially available, and even when their metabolic pathways are known, their biosynthesis, purification, and in vitro analysis is a resource-intensive process that is difficult to parallelize with existing methods 54 . Our B2H system offers a potential solution: It can identify inhibitor-synthesizing genes with a simple growth-coupled assay. We explored its application to discovery efforts by using it to screen a diverse set of uncharacterized biosynthetic genes. In brief, we carried out a bioinformatic analysis of the largest terpene synthase family (PF03936) by building and annotating a cladogram of its 4,464 constituent members ( FIG.  27   ); from here, we synthesized three uncharacterized genes from each of eight clades: six with no characterized genes and two with some characterized genes ( FIG.  24 A ). We reasoned that these 24 phylogenetically diverse genes (8 from fungi, 13 from plants, and 3 from bacteria) might encode enzymes with distinct product profiles and potentially, through the inclusion of uncharacterized clades, novel sesquiterpene scaffolds. 
     Guided by our initial screen, we searched for sesquiterpene inhibitors by pairing each of the uncharacterized genes with the FPP pathway. To our surprise, six genes conferred a significant survival advantage ( FIG.  24 B ), and maximal resistance required an active B2H system ( FIG.  28   ). Each hit generated distinct product profiles ( FIG.  29   ); we focused our analysis on A0A0C9VSL7, which produced mostly (+)-1(10),4-cadinadiene as a major product ( FIGS.  24 C- 24 D ). This terpenoid is a structural analog of AD but has a weaker potency (IC 50 =165±33 μM;  FIG.  24 E ); a titer of 33±18 μM suggests that intracellular accumulation may allow it to inhibit PTP1B inside the cell. Our ability to detect a weak inhibitor suggests that the B2H system can capture a broad set of scaffolds in molecular discovery efforts. The purification and analysis of additional hits, the incorporation of isoprenoid substrates of different sizes (through the use of geranyl diphosphate synthase or geranyl geranyl diphosphate synthase), and the inclusion of more uncharacterized genes could expand the scope of such efforts. 
     Design of Alternative PTP-Specific Objectives 
     We explored the versatility of our B2H system by assessing its ability to detect the inactivation of several other diseases-relevant PTPs. In short, we swapped out the gene for PTP1B with genes for PTPN2, PTPN6, or PTPN12; these enzymes are targets for immunotherapeutic enhancement 55 , the treatment of ovarian cancer 56 , and acute myocardial infarction 57 , respectively. Their catalytic domains share 31-65% sequence identity with the catalytic domain of PTP1B. Interestingly, the new B2H systems were immediately functional; PTP inactivation permitted growth at high concentrations of spectinomycin ( FIG.  25 A ). This finding suggests that our detection system can be easily extended to other members of the PTP family. 
     PTP-specific B2H systems could facilitate the identification of natural products that selectively inhibit one PTP over another. We explored this application by comparing the antibiotic resistance conferred by PTP1B- and TC-PTP-specific systems in response to metabolic pathways for AD and α-bisabolene ( FIG.  25 B ). As expected, the PTP1B-specific system permitted growth at higher concentrations of antibiotic, a result consistent with the selectivity of both terpenoids for PTP1B. Indistinguishable terpenoid titers between the two strains suggest that this survival advantage does not result from difference in intracellular concentration ( FIG.  25 C ). Findings thus indicate that a simple comparison of B2H systems—a potential secondary screen—offers a simple approach for evaluating the selectivity PTP-inhibiting gene products. Notably, high concentrations of inhibitors in two strains could swamp out selective effects; in such cases, terpenoid levels could be reduced with lower mevalonate concentrations. 
     This study addresses an important challenge of medicinal chemistry—the design of molecular structures that inhibit disease-relevant enzymes—by using a desired biochemical activity (i.e., an objective) as a genetically encoded constraint to guide molecular biosynthesis. This approach enabled the identification of two selective, biologically active inhibitors of PTP1B, an elusive drug target 58 . These molecules are not drugs, but they are promising scaffolds for lead development. Their mechanisms of modulation—which elicit allosteric conformational changes yet appear to rely on loose, conformationally flexible binding—are unusual (and computationally elusive 59 ), and demonstrate the ability of microbial systems to find new solutions to difficult challenges in molecular design. Our identification of unusual inhibitors in relatively small libraries, in turn, suggests that microbial systems can access a rich molecular landscape that is not efficiently explored by existing approaches to molecular discovery. 
     The B2H system at the core of our approach is a valuable tool for identifying biologically active natural products, which are structurally complex, difficult to synthesize, and often hidden in cryptic gene clusters 60 . It has several key advantages over contemporary approaches to inhibitor discovery: (i) It incorporates synthesizability as a search criterion—an important attribute of drug leads 61 . (ii) It is scalable. We used a growth-coupled assay to screen 24 uncharacterized terpene synthases; this type of assay is also compatible with very large mutagenesis libraries (e.g., 1010) 62 . (iii) It can use cellular machinery to stabilize proteins (e.g., CDC37 for Src); this capability could facilitate the integration of unstable and/or disordered targets. Future efforts to exploit these advantages by incorporating large libraries of mutated and/or reconfigured pathways, alternative biosynthetic enzymes (e.g., cytochromes P450, halogenases, and methyltransferases), or new classes of disease-relevant enzymes would be informative. 
     The B2H system also has important limits. When used alongside metabolic pathways, it links survival not only to the potency of metabolites, but also to their titers, off-target effects, and pathway toxicities. These limitations can be beneficial; they bias the discovery process toward potent, readily synthesizable inhibitors and could, thus, facilitate post-discovery efforts to improve the titers of interesting molecules 63 . Nonetheless, they will exclude some types of structurally complex molecules that are difficult to synthesize in  E. coli . The use of similar activity-based screens in other organisms (e.g.,  Streptomyces ) could be interesting. 
     The compatibility of our discovery approach with different PTPs is valuable in light of their increasingly well validated potential as a rich—and essentially untapped—source of new therapeutic targets 64 . We anticipate that some PTPs will require the use of chaperones and/or transcriptional adjustments to be incorporated into B2H systems. Our systematic optimization of the PTP1B-based system provides an experimental framework for exploring these modifications. Side-by-side comparisons of B2H systems, in turn, offer a promising strategy for evaluating inhibitor selectivity in secondary screens. In future work, new varieties of objectives (e.g., B2H systems or genetic circuits that detect the selective inhibition—or, perhaps, activation—of one PTP over another) could facilitate the discovery of molecules with sophisticated mechanisms of modulation in primary screens. The versatility of genetically encoded objectives highlights the power of using microbial systems to find targeted, biologically active molecules. 
     Note 1: The orthogonality of proteomes.  E. coli  and  S. cerevisiae  are both well-developed platforms for the production of pharmaceutically relevant natural products 20,65,66 . We chose to use  E. coli  for this study because its machinery for phosphorylating proteins is dissimilar from that of eukaryotic cells and thus less likely to interfere with the function of genetically encoded systems that link the inhibition of PTP1B to cellular growth 67 . By contrast, the overexpression of Src kinase in  S. cerevisiae  is lethal and is mitigated by PTP1B 68 ; these effects are inconsistent with our biochemical objective. More broadly,  S. cerevisiae  and humans, despite having evolved from a common ancestor approximately 1 billion years ago 69 , share many functionally equivalent proteins; orthologous genes, in fact, account for more than one-third of the yeast genome 70 . Most strikingly, a recent study found that nearly half (47%) of 414 essential genes from  S. cerevisiae  could be replaced with human orthologs without growth defects 71 . This finding suggests that yeast is a particularly restrictive host for genetically encoded systems that link arbitrary changes in the activities of human regulatory enzymes to fitness advantage. 
     Methods 
     Bacterial strains. We used  E. coli  DH10B, chemically competent NEB Turbo, or electrocompetent One Shot Top10 (Invitrogen) to carry out molecular cloning and to perform preliminary analyses of terpenoid production; we used  E. coli  BL2-DE31 to express proteins for in vitro studies; and we used  E. coli  s1030 72  for our luminescence studies and for all experiments involving terpenoid-mediated growth (i.e., evolution studies). 
     For all strains, we generated chemically competent cells by carrying out the following steps: (i) We plated each strain on LB agar plates with the required antibiotics. (ii) We used one colony of each strain to inoculate 1 mL of LB media (25 g/L LB with appropriate antibiotics listed in TABLE 8) in a glass culture tube, and we grew this culture overnight (37° C., 225 RPM). (iii) We used the 1-mL culture to inoculate 100-300 mL of LB media (as above) in a glass shake flask, and we grew this culture for several hours (37° C., 225 RPM). (iv) When the culture reached an OD of 0.3-0.6, we centrifuged the cells (4,000×g for 10 minutes at 4° C.), removed the supernatant, resuspended them in 30 mL of ice cold TFB1 buffer (30 mM potassium acetate, 10 mM CaCl 2 , 50 mM MnCl 2 , 100 mM RbCl, 15% v/v glycerol, water to 200 mL, pH=5.8, sterile filtered), and incubated the suspension at 4° C. for 90 min. (v) We repeated step iv, but resuspended in 4 mL of ice cold TFB2 buffer (10 mM MOPS, 75 mM CaCl 2 , 10 mM RbCl 2 , 15% glycerol, water to 50 mL, pH=6.5, sterile filtered). (iv) We split the final suspension into 100 μL aliquots and froze them at −80° C. until further use. 
     We generated electrocompetent cells by following an approach similar to the one above. In step iv, however, we resuspended the cells in 50 mL of ice cold MilliQ water and repeated this step twice—first with 50 mL of 20% sterile glycerol (ice cold) and, then, with 1 mL of 20% sterile glycerol (ice cold). We froze the pellets as before. 
     Materials. We purchased methyl abietate from Santa Cruz Biotechnology; trans-caryophyllene, tris(2-carboxyethyl)phosphine (TCEP), bovine serum albumin (BSA), M9 minimal salts, phenylmethylsulfonyl fluoride (PMSF), and DMSO (dimethyl sulfoxide) from Millipore Sigma; glycerol, bacterial protein extraction reagent II (B-PERII), and lysozyme from VWR; cloning reagents from New England Biolabs; AD from Ambeed, Inc.; and all other reagents (e.g., antibiotics and media components) from Thermo Fisher. Taxadiene was a kind gift from Phil Baran of the The Scripps Research Institute. We prepared mevalonate by mixing 1 volume of 2 M DL-mevalanolactone with 1.05 volumes of 2 M KOH and incubating this mixture at 37° C. for 30 minutes.
 
Cloning and molecular biology. We constructed all plasmids by using standard methods (i.e., restriction digest and ligation, Golden Gate and Gibson assembly, Quikchange mutagenesis, and circular polymerase extension cloning). TABLE 7 describes the source of each gene; TABLE 8 and TABLE 3 describe the composition of all final plasmids.
 
     We began construction of the B2H system by integrating the gene for HA4-RpoZ from pAB094a into pAB078d and by replacing the ampicillin resistance marker of pAB078d with a kanamycin resistance marker (Gibson Assembly). We modified the resulting “combined” plasmid, in turn, by replacing the HA4 and SH2 domains with kinase substrate and substrate recognition (i.e., SH2) domains, respectively (Gibson assembly), and by integrating genes for Src kinase, CDC37, and PTP1B in various combinations (Gibson assembly). We finalized the functional B2H system by modifying the SH2 domain with several mutations known to enhance its affinity for phosphopeptides (K15L, T8V, and C10A, numbered as in Kaneko et. al. 35 ), by exchanging the GOI for luminescence (LuxAB) with one for spectinomycin resistance (SpecR), and by toggling promoters and ribosome binding sites to enhance the transcriptional response (Gibson assembly and Quickchange Mutagenesis, Agilent Inc.). We note: For the last step, we also converted Prol to ProD by using the Quikchange protocol. When necessary, we constructed plasmids with arabinose-inducible components by cloning a single component from the B2H system into pBAD (Golden Gate assembly). TABLE 4, TABLE 9, and TABLE 10 list the primers and DNA fragments used to construct each plasmid. 
     We assembled pathways for terpenoid biosynthesis by purchasing plasmids encoding the first module (pMBIS) and various sesquiterpene synthases (ADS or GHS in pTRC99a) from Addgene, and by building the remaining plasmids. We replaced the tetracycline resistance in pMBIS with a gene for chloramphenicol resistance to create pMBIS CmR . We integrated genes for ABS, TXS, ABA, and GGPPS into pTRC99t (i.e., pTRC99a without BsaI sites). TABLE 4, TABLE 9, and TABLE 10 list the primers and DNA fragments used to construct each plasmid. 
     Luminescence assays. We characterized preliminary B2H systems (which contained LuxAB as the GOI) with luminescence assays. In brief, we transformed necessary plasmids into  E. coli  s1030 (TABLE 8), plated the transformed cells onto LB agar plates (20 g/L agar, 10 g/L tryptone, 10 g/L sodium chloride, and 5 g/L yeast extract with antibiotics described in TABLE 8), and incubated all plates overnight at 37° C. We used individual colonies to inoculate 1 ml of terrific both (TB at 2%, or 12 g/L tryptone, 24 g/L yeast extract, 12 mL/L 100% glycerol, 2.28 g/L KH 2 PO 4 , 12.53 g/L K 2 HPO 4 , pH=7.3, and antibiotics described in TABLE 8), and we incubated these cultures overnight (37° C. and 225 RPM). The following morning, we diluted each culture by 100-fold into 1 ml of TB media (above), and we incubated these cultures in individual wells of a deep 96-well plate for 5.5 hours (37° C., 225 RPM). (We note: When pBAD was present, we supplemented the TB media with 0-0.02 w/v % arabinose). We transferred 100 μL of each culture into a single well of a standard 96-well clear plate and measured both OD 600  and luminescence on a Biotek Synergy plate reader (gain: 135, integration time: 1 second, read height: 1 mm). Analogous measurements of cell-free media allowed us to measure background signals, which we subtracted from each measurement prior to calculating OD-normalized luminescence (i.e., Lum/OD 600 ).
 
Analysis of antibiotic resistance. We evaluated the spectinomycin resistance conferred by various B2H systems in the absence of terpenoid pathways by carrying out the following steps: (i) We transformed  E. coli  with the necessary plasmids (TABLE 8) and plated the transformed cells onto LB agar plates (20 g/L agar, 10 g/L tryptone, 10 g/L sodium chloride, 5 g/L yeast extract, 50 μg/ml kanamycin, 10 μg/ml tetracycline). (ii) We used individual colonies to inoculate 1-2 ml of TB media (12 g/L tryptone, 24 g/L yeast extract, 12 mL/L 100% glycerol, 2.28 g/L KH 2 PO 4 , 12.53 g/L K 2 HPO 4 , 50 μg/ml kanamycin, 10 μg/ml tetracycline, pH=7.3), and we incubated these cultures overnight (37° C., 225 RPM). In the morning, we diluted each culture by 100-fold into 4 ml of TB media (as above) with 0-500 μg/ml spectinomycin (we used spectinomycin in the liquid culture only for  FIG.  14   ), and we incubated these cultures in deep 24-well plates until wells containing 0 μg/ml spectinomycin reached an OD 600  of 0.9-1.1. (iv) We diluted each 4-ml culture by 10-fold into TB media with no antibiotics and plated 10-μL drops of the diluent onto agar plates with various concentrations of spectinomycin. (v) We incubated plates overnight (37° C.) and photographed them the following day.
 
     To examine terpenoid-mediated resistance, we began with steps i and ii as described above with the addition of 34 μg/ml chloramphenicol and 50 μg/ml carbenicillin in all liquid/solid media. We then proceeded with the following steps: (iii) We diluted samples from 1-ml cultures to an OD 600  of 0.05 in 4.5 ml of TB media (supplemented with 12 g/L tryptone, 24 g/L yeast extract, 12 mL/L 100% glycerol, 2.28 g/L KH 2 PO 4 , 12.53 g/L K 2 HPO 4 , 50 μg/ml kanamycin, 10 μg/ml tetracycline, 34 μg/ml chloramphenicol, and 50 μg/ml carbenicillin), which we incubated in deep 24-well plates (37° C., 225 RPM). (iv) At an OD 600  of 0.3-0.6, we transferred 4 ml of each culture to a new well of a deep 24-well plate, added 500 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) and 20 mM of mevalonate, and incubated for 20 hours (22° C., 225 RPM). (v) We diluted each 4-ml culture to an OD 600  of 0.1 with TB media and plated 10 μL of the diluent onto either LB or TB plates supplemented with 500 μM IPTG, 20 mM mevalonate, 50 μg/ml kanamycin, 10 μg/ml tetracycline, 34 μg/ml chloramphenicol, 50 μg/ml carbenicillin, and 0-1200 μg/ml spectinomycin (for both plates, we used 20 g/L agar with media and buffer components described above). 
     Terpenoid biosynthesis. We prepared  E. coli  for terpenoid production by transforming cells with plasmids harboring requisite pathway components (TABLE 8) and plating them onto LB agar plates (20 g/L agar, 10 g/L tryptone, 10 g/L sodium chloride, and 5 g/L yeast extract with antibiotics described in TABLE 8). We used one colony from each strain to inoculate 2 ml TB (12 g/L tryptone, 24 g/L yeast extract, 12 mL/L 100% glycerol, 2.28 g/L KH 2 PO 4 , 12.53 g/L K 2 HPO 4 , pH=7.0, and antibiotics described in TABLE 8) in a glass culture tube for ˜16 hours (37° C. and 225 RPM). We diluted these cultures by 75-fold into 10 ml of TB media and incubated the new cultures in 125 mL glass shake flasks (37° C. and 225 RPM). At an OD 600  of 0.3-0.6, we added 500 μM IPTG and 20 mM mevalonate. After 72-88 hours of growth (22° C. and 225 RPM), we extracted terpenoids from each culture as outlined below.
 
Protein expression and purification. We expressed and purified PTPs as described previously 73 . Briefly, we transformed  E. coli  BL21(DE3) cells with pET16b or pET21b vectors (see TABLE 8 for details), and we induced with 500 μM IPTG at 22° C. for 20 hours. We purified PTPs from cell lysate by using desalting, nickel affinity, and anion exchange chromatography (HiPrep 26/10, HisTrap HP, and HiPrep Q HP, respectively; GE Healthcare). We stored the final protein (30-50 μM) in HEPES buffer (50 mM, pH 7.5, 0.5 mM TCEP) in 20% glycerol at ˜80° C.
 
Extraction and purification of terpenoids. We used hexane to extract terpenoids generated in liquid culture. For 10-mL cultures, we added 14 mL of hexane to 10 ml of culture broth in 125-mL glass shake flasks, shook the mixture (100 RPM) for 30 minutes, centrifuged it (4000×g), and withdrew 10 mL of the hexane layer for further analysis. For 4-mL cultures, we added 600 μL hexane to 1 mL of culture broth in a microcentrifuge tube, vortexed the tubes for 3 minutes, centrifuged the tubes for 1 minute (17000×g), and saved 300-400 μL of the hexane layer for further analysis.
 
     To purify AD, AB, and (+)-1(10),4-cadinadiene, we supplemented 500-1000 mL culture broth with hexane (16.7% v/v), shook the mixture for 30 minutes (100 RPM), isolated the hexane layer with a separatory funnel, centrifuged the isolated organic phase (4000×g), and withdrew the hexane layer. To concentrate the terpenoid products, we evaporated excess hexane in a rotary evaporator to bring the final volume to 500 μL, and we passed the resulting mixture over a silica gel 1-3 times (Sigma-Aldrich; high purity grade, 60 Å pore size, 230-400 mesh particle size). We analyzed elution fractions (100% hexane) on the GC/MS and pooled fractions with the compound of interest (AD). Once purified, we dried pooled fractions under a gentle stream of air, resuspended the concentrated terpenoids in DMSO, and quantified the final samples as outlined below. We repeated the purification process until samples (in DMSO) were &gt;95% pure by GC/MS unless otherwise noted. 
     GC-MS analysis of terpenoids. We measured terpenoids generated in liquid culture with a gas chromatograph/mass spectrometer (GC-MS; a Trace 1310 GC fitted with a TG5-SilMS column and an ISQ 7000 MS; Thermo Fisher Scientific). We prepared all samples in hexane (directly or through a 1:100 dilution of DMSO) with 20 μg/ml of caryophyllene as an internal standard. Highly concentrated samples were diluted 10-20× prior to preparation to bring concentrations within the MS detection limit. When the peak area of an internal standard exceeded ±40% of the average area of all samples containing that standard, we re-analyzed the corresponding samples. For all runs, we used the following GC method: hold at 80° C. (3 min), increase to 250° C. (15° C./min), hold at 250° C. (6 min), increase to 280° C. (30° C./min), and hold at 280° C. (3 min). To identify various analytes, we scanned m/z ratios from 50 to 550. 
     We examined sesquiterpenes generated by variants of ADS by using select ion mode (SIM) to scan for the molecular ion (m/z=204). For quantification, we used Eq. 1: where A i   
     
       
         
           
             
               
                 
                   
                     C 
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                       C 
                       std 
                     
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                     R 
                   
                 
               
               
                 
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                         A 
                         
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                         A 
                         
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     is the area of the peak produced by analyte i, A std  is the area of the peak produced by C std  of caryophyllene in the sample, and R is the ratio of response factors for caryophyllene and AD in a reference sample. TABLE 11 provides the concentrations of all standards and reference compounds used in this analysis. 
     We quantified diterpenoids by, once again, accompanying our general procedure with several modifications: We scanned for a different molecular ion (m/z=272) and an ion common to both diterpenoids and caryophyllene (m/z=93); we used a ratio of response factors for pure taxadiene (a kind gift from Phil Baran) and caryophyllene at m/z=93; and we calculated peak areas m/z=93. For all analyses, we examined only peaks with areas that exceeded 1% of the total area of all peaks at m/z=272. 
     We identified molecules by using the NIST MS library and, when necessary, confirmed this identification with analytical standards or mass spectra reported in the literature. We note: The assumption of a constant response factor for different terpenoids (that is, the assumption that all sesquiterpenes and diterpenes ionize like AD and taxadiene, respectively) can certainly yield error in estimates of their concentrations; our analyses, which are consistent with those of other studies of terpenoid production in microbial systems 74,75 , supply rough estimates of concentrations for all compounds except AD and taxadiene (which had analytical standards). 
     Bioinformatics. We used a bioinformatic analysis to identify a phylogenetically diverse set of terpene synthases. Briefly, we downloaded (i) all constituent genes of PF03936 (the largest terpene synthase family grouped by a C-terminal domain) from the PFAM Database and (ii) all enzymes with Enzyme Commission (EC) number of 4.2.3.# from the Uniprot Database; this string, which defines carbon oxygen lyases that act on phosphates, includes terpene synthases. We cleaned both datasets in Excel (i.e., we ensured that every identifier had only one row), and we used a custom R script to designate each PF03936 member as characterized (i.e., in possession of a Uniprot-based EC number) or uncharacterized. Finally, we used FastTree 76  with default settings to create a phylogenetic tree of the PF03936 family and the R-package ggtree 77  to visualize the resulting tree and function data as a cladogram and heatmap. 
     After annotating the cladogram by hand, we selected three genes from each of six clades: six with no characterized genes and two with some characterized genes. We avoided clades proximal to known monoterpene synthases or diterpene synthases known to act on GGPP isomers absent in our system (e.g., ent-copalyl diphosphate); these enzymes are unlikely to act on FPP, the primary product of pMBIS CmR . When selecting enzymes within clades, we biased our choice towards bacterial/fungal species and selected genes with a minimal number of common ancestors within the Glade. The selected genes were synthesized and cloned into the pTrc99a vector by Twist Biosciences and assayed for antibiotic resistance as described above. 
     Enzyme kinetics. To examine terpenoid-mediated inhibition, we measured PTP-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) or 4-methylumbelliferyl phosphate (4-MUP, used when KM for pNPP was large) in the presence of various concentrations of terpenoids. Each reaction included PTP (0.05 μM PTP1B/TCPTP or 0.1 μM SHP1/SHP2 in 50 mM HEPES, 0.5 mM TCEP, 50 μg/ml BSA), pNPP (0.33, 0.67, 2, 5, 10, and 15 mM) or 4-MUP (0.13, 0.27, 0.8, 2.27, 2.93, 4.53, 7.07, and 8 mM), inhibitor (with concentrations listed in the figures), buffer (50 mM HEPES pH=7.3, 50 μg/ml BSA), and DMSO at 10% v/v. We monitored the formation of p-nitrophenol by measuring absorbance at 405 nm every 10 seconds for 5 minutes on a SpectraMax M2 plate reader and the formation of 4-methylumbelliferyl by measuring fluorescence at 450 nm (370 nm ex, 435 nm cutoff, medium gain). 
     We used a custom MATLAB script to process all raw kinetic data. This script removed all concentration values that fell outside of either (i) the range of our standard curve (absorbance/fluorescence vs. μM;  FIG.  39   ) or (ii) the initial rate regime (&gt;10% of the pNPP or 4-MUP concentration used in the assay). When this step reduced kinetic dataset to fewer than ten points, we re-measured those datasets to collect at least ten. We fit final datasets, in turn, with a linear regression model (using Matlab&#39;s backslash operator). 
     We evaluated kinetic models in three steps: (i) We fit initial-rate measurements collected in the absence and presence of inhibitors to Michaelis-Menten and inhibition models, respectively (here, we used the nlinfit and fminsearch functions from MATLAB; TABLE 12). (ii) We used an F-test to compare the mixed model to the single-parameter model with the least sum squared error (here, we used the fcdf function from MATLAB to assign p-values), and we accepted the mixed model when p&lt;0.05. (iii) We used the Akaike&#39;s Information Criterion (AIC) to compare the best-fit single parameter model to each alternative single parameter model, and we accepted the “best-fit” model when the difference in AIC (Δ i ) exceed 5 for all comparisons. 78  We note: For AD, AB, and (+)1-(10),4-cadinadiene this criterion was not met; both noncompetitive and uncompetitive models, however, yielded indistinguishable IC 50 &#39;s. 
     We estimated the half maximal inhibitory concentration (IC 50 ) of inhibitors by using the best-fit kinetic models to determine the concentration of inhibitor required to reduce initial rates of PTP-catalyzed hydrolysis of 15 mM of pNPP by 50%. We used the MATLAB function “nlparci” to determine the confidence intervals of kinetic parameters, and we propagated those intervals to estimate corresponding confidence intervals for each IC 50 . 
     X-ray crystallography. We prepared crystals of PTP1B by using hanging drop vapor diffusion. In brief, we added 2 μL of PTP1B (˜600 μM PTP1B, 50 mM HEPES, pH 7.3) to 6 μL of crystallization solution (100 mM HEPES, 200 mM magnesium acetate, and 14% polyethylene glycol 8000, pH 7.5) and incubated the resulting droplets over crystallization solution for one week at 4° C. (EasyXtal CrystalSupport, Qiagen). We soaked crystals with ligand by transferring them to droplets formed with 6 μL of crystallization solution and 1 μL of ligand solution (10 mM in DMSO), which we incubated for 2-5 days at 4° C. We prepared all ligands for freezing by soaking them in cryoprotectant formed from a 70/30 (v/v) mixture of buffer (100 mM HEPES, 200 mM magnesium acetate, and 25% polyethylene glycol 8000, pH 7.5) and glycerol. 
     We collected X-ray diffraction data through the Collaborative Crystallography Program at Lawrence Berkeley National Lab (ALS ENABLE, beamline 8.2.1, 100 K, 1.00003 Å). We performed integration, scaling, and merging of X-ray diffraction data using the xia2 software package 79 , and we carried out molecular replacement and structure refinement with the PHENIX graphical interface, 80  supplemented with manual model adjustment in COOT 81  and one round of PDB-REDO 82  (the latter, only for the PTP1B-AD complex). 
     Molecular dynamics (MD) simulations. Full-length PTP1B contains a disordered region that extends beyond the α7 helix (i.e., 299-435). In this study, we used a well-studied truncation variant (i.e., PTP1B 1-321 ) that includes residues from the disordered region. To model PTP1B, we used CAMPARI v.2 83  to generate structures of the disordered region of each complex (i.e., residues 288-321 for PTP1B-AD) from a crystal structure without a disordered tail. To quickly thermalize the tail structures, we ran short Monte Carlo (MC) simulations using the ABSINTH implicit-solvent force field 84,85 , fixing the coordinates of the atoms in the ligand and the protein core. 
     We performed MD simulations using GROMACS 2020 86 . Briefly, we used the CHARMM36m protein force field 87 , a CHARMM-modified TIP3P water model 88 , and ligand parameters generated by CGenFF 89,90 . We solvated each PTP1B-ligand complex (initialized from the corresponding crystal structure) in a dodecahedral box with edges positioned ≥10 Å from the surface of the complex, and we added six sodium ions to neutralize each system. We used the LINCS algorithm 91  to constrain all bonds involving hydrogen atoms, the Verlet leapfrog algorithm to numerically integrate equations of motion with a 2-fs time step, and the particle-mesh Ewald summation 92  (cubic interpolation with a grid spacing of 0.16 nm) to calculate long-range electrostatic interactions; we used a cutoff of 1.2 nm, in turn, for short-range electrostatic and Lennard-Jones interactions. We independently coupled the protein-ligand complex and solvent molecules to a temperature bath (300K) using a modified Berendsen thermostat 93  with a relaxation time of 0.1 ps, and we fixed pressure coupling to 1 bar using the Parrinello-Rahman algorithm 94  with a relaxation time of 2 ps and isothermal compressibility of 4.5×10 −5  bar −1 . 
     For each system, we carried out 30 independent MD simulations to reduce sampling bias. For each MD trajectory, we minimized energy using the steepest decent method followed by 100-ps solvent relaxation in the NVT ensemble and 100-ps solvent relaxation in the NPT ensemble. After an additional 5-ns NPT equilibration, we carried out production runs for 5 ns in the NPT ensemble and registered coordinate data every 10 ps. 
     Analysis of PTP1B inhibition in HEK293TCells. We prepared HEK293T/17 cells for an enzyme-linked immunosorbent assay (ELISA) by growing them in 75 cm 2  culture flasks (Corning) with DMEM media supplemented with 10% FBS, 100 units/ml penicillin, and 100 units/ml streptomycin. We replaced the media every day for 3-5 days until the cells reached 80-100% confluency. 
     We measured the influence of inhibitors on insulin receptor (IR) phosphorylation by using an IR-specific ELISA ( FIG.  35   ). Briefly, we starved cells for 48 hours in FBS-free media and incubated the with inhibitors (all at 3% DMSO) for 10 minutes. After incubation, we lysed cells with lysis buffer (9803, Cell Signaling Technology) supplemented with 1× halt phosphatase inhibitor cocktail and 1× halt protease inhibitor cocktail (Thermo Fisher Scientific) for 10 min, pelleted the cell debris, and used the lysis buffer to dilute each sample to 60 mg/ml total protein. We measured IR phosphorylation in subsequent dilutions of the 60 mg/ml samples with the PathScan® Phospho-Insulin Receptor β (panTyr) Sandwich ELISA Kit (Cell Signaling Technology; #7082). We note: To identify biologically active concentrations of AB and AD, we screened several concentrations and chose those that gave the highest signal (405 μM for AB and 930 μM for AD); similar concentrations of weak inhibitors did not yield a detectable signal ( FIGS.  35 B and  35 C ). 
     Statistical analysis and reproducibility. We determined statistical significance ( FIG.  23 H ) with a two-tailed Student&#39;s t-test (details in TABLE 14), and we used an F-test to compare one- and two-parameter models of inhibition (TABLE 12). 
     REFERENCES FOR EXAMPLE 2 
     
         
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         48. Vallurupalli, P., Bouvignies, G. &amp; Kay, L. E. Studying ‘invisible’ excited protein states in slow exchange with a major state conformation.  J. Am. Chem. Soc.  134, 8148-8161 (2012). 
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         51. Goldstein, B. J., Bittner-Kowalczyk, A., White, M. F. &amp; Harbeck, M. Tyrosine dephosphorylation and deactivation of insulin receptor substrate-1 by protein-tyrosine phosphatase 1B. Possible facilitation by the formation of a ternary complex with the GRB2 adaptor protein.  J. Biol. Chem.  275, 4283-4289 (2000). 
         52. Choi, E. et al. Mitotic regulators and the SHP2-MAPK pathway promote IR endocytosis and feedback regulation of insulin signaling.  Nat. Commun.  10, (2019). 
         53. Dubois, M. J. et al. The SHP-1 protein tyrosine phosphatase negatively modulates glucose homeostasis.  Nat. Med.  12,549-556 (2006). 
         54. Hubert, J., Nuzillard, J. M. &amp; Renault, J. H. Dereplication strategies in natural product research: How many tools and methodologies behind the same concept?  Phytochemistry Reviews  16, 55-95 (2017). 
         55. Manguso, R. T. et al. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target.  Nature  547, 413-418 (2017). 
         56. Varone, A., Spano, D. &amp; Corda, D. Shpl in Solid Cancers and Their Therapy.  Frontiers in Oncology  10, 935 (2020). 
         57. Yang, C. F. et al. Targeting protein tyrosine phosphatase PTP-PEST (PTPN12) for therapeutic intervention in acute myocardial infarction.  Cardiovasc. Res.  116, 1032-1046 (2020). 
         58. Zhang, S. &amp; Zhang, Z. Y. PTP1B as a drug target: recent developments in PTP1B inhibitor discovery.  Drug Discov. Today  12, 373-381 (2007). 
         59. Oleinikovas, V., Saladino, G., Cossins, B. P. &amp; Gervasio, F. L. Understanding Cryptic Pocket Formation in Protein Targets by Enhanced Sampling Simulations.  J. Am. Chem. Soc.  138, 14257-14263 (2016). 
         60. Rutledge, P. J. &amp; Challis, G. L. Discovery of microbial natural products by activation of silent biosynthetic gene clusters.  Nature Reviews Microbiology  13, 509-523 (2015). 
         61. Hartenfeller, M. &amp; Schneider, G. De Novo Drug Design. in  Chemoinformatics and Computational Chemical Biology  (ed. Bajorath, J.) 299-323 (Humana Press, 2011). doi:10.1007/978-1-60761-839-3_12 
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         63. Johnston, C. W., Badran, A. H. &amp; Collins, J. J. Continuous bioactivity-dependent evolution of an antibiotic biosynthetic pathway.  Nat. Commun.  11, 4202 (2020). 
         64. Chen, M. J., Dixon, J. E. &amp; Manning, G. Genomics and evolution of protein phosphatases.  Sci. Signal.  10, 1-17 (2017). 
         65. Galanie, S. et al. Complete biosynthesis of opioids in yeast.  Science  (80-.). 349,1095-1100 (2015). 
         66. Paddon, C. J. &amp; Keasling, J. D. Semi-synthetic artemisinin: a model for the use of synthetic biology in pharmaceutical development.  Nat. Rev. Microbiol.  12,355-367 (2014). 
         67. Grangeasse, C., Nessler, S. &amp; Mijakovic, I. Bacterial tyrosine kinases: Evolution, biological function and structural insights.  Philos. Trans. R. Soc. B Biol. Sci.  367, 2640-2655 (2012). 
         68. Montalibet, J. et al. Residues distant from the active site influence protein-tyrosine phosphatase 1B inhibitor binding.  J. Biol. Chem.  281,5258-5266 (2006). 
         69. Douzery, E. J. P., Snell, E. A., Bapteste, E., Delsuc, F. &amp; Philippe, H. The timing of eukaryotic evolution: Does a relaxed molecular clock reconcile proteins and fossils?  Proc. Natl. Acad. Sci. U.S.A.  101, 15386-15391 (2004). 
         70. O&#39;Brien, K. P., Remm, M. &amp; Sonnhammer, E. L. L. Inparanoid: A comprehensive database of eukaryotic orthologs.  Nucleic Acids Res.  33, D476-D480 (2005). 
         71. Kachroo, A. H. et al. Systematic humanization of yeast genes reveals conserved functions and genetic modularity.  Science  (80-.). 348, 921-925 (2015). 
         72. Carlson, J. C., Badran, A. H., Guggiana-Nilo, D. A. &amp; Liu, D. R. Negative selection and stringency modulation in phage-assisted continuous evolution.  Nat. Chem. Biol.  10, 216-222 (2014). 
         73. Hjortness, M. K. et al. Evolutionarily Conserved Allosteric Communication in Protein Tyrosine Phosphatases.  Biochemistry  57, 6443-6451 (2018). 
         74. Chen, X. et al. Statistical experimental design guided optimization of a one-pot biphasic multienzyme total synthesis of amorpha-4,11-diene.  PLoS One  8, e79650 (2013). 
         75. Edgar, S. et al. Mechanistic Insights into Taxadiene Epoxidation by Taxadiene-5α-Hydroxylase.  ACS Chem. Biol.  11, 460-469 (2016). 
         76. Price, M. N., Dehal, P. S. &amp; Arkin, A. P. FastTree 2—Approximately maximum-likelihood trees for large alignments.  PLoS One  5, e9490 (2010). 
         77. Yu, G., Smith, D. K., Zhu, H., Guan, Y. &amp; Lam, T. T. Y. ggtree: an r package for visualization and annotation of phylogenetic trees with their covariates and other associated data.  Methods Ecol. Evol.  8, 28-36 (2017). 
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     Tables 
       
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Gene Sources 
               
            
           
           
               
               
               
               
            
               
                 Component 
                 Organism 
                 Plasmid 
                 Source 
               
               
                   
               
               
                 Src 
                 
                   H. sapiens 
                 
                 pDONR223_ 
                 Addgene: 82165 
               
               
                   
                   
                 SRC_WT 
                   
               
               
                 CDC37 
                 
                   H. sapiens 
                 
                 pBACgus4x/ 
                 Addgene: 40398 
               
               
                   
                   
                 cdc37/ 
                   
               
               
                   
                   
                 RocCOR 
                   
               
               
                   
                   
                 LRRK2  
                   
               
               
                   
                   
                 1867-2176 
                   
               
               
                 PTP1B 
                 
                   H. sapiens 
                 
                 pGEX-2T  
                 Addgene: 8602 
               
               
                   
                   
                 PTP-1B 
                   
               
               
                 SHP2 
                 
                   H. sapiens 
                 
                 PTPN11 
                 Addgene: 38965 
               
               
                 TC-PTP 
                 
                   H. sapiens 
                 
                 pBG100- 
                 Addgene: 33365 
               
               
                   
                   
                 TCPTP 
                   
               
               
                 LuxAB 
                   
                 pAB078d8 
                 Addgene: 79206 
               
               
                 RpoZ 
                 
                   Escherichia  
                 
                 pAB094a 
                 Addgene: 79241 
               
               
                   
                 
                   coli 
                 
                   
                   
               
               
                 cI434 
                 
                   Escherichia  
                 
                 pAB078d8 
                 Addgene: 79206 
               
               
                   
                 
                   virus 
                 
                   
                   
               
               
                   
                 
                   Lambda 
                 
                   
                   
               
               
                 SH2 
                 
                   Rous  
                 
                   
                 Addgene: 78302 
               
               
                   
                 
                   sarcoma 
                 
                   
                   
               
               
                   
                 
                   virus 
                 
                   
                   
               
               
                 p130cas 
                 
                   H. sapiens 
                 
                 Synthetic 
                 Integrated DNA  
               
               
                   
                   
                   
                 Technologies, Inc. 
               
               
                 midT 
                 
                   H. sapiens 
                 
                 Synthetic 
                 Integrated DNA  
               
               
                   
                   
                   
                 Technologies, Inc. 
               
               
                 EGFR 
                 
                   H. sapiens 
                 
                 Synthetic 
                 Integrated DNA  
               
               
                   
                   
                   
                 Technologies, Inc. 
               
               
                 ShcA 
                 
                   H. sapiens 
                 
                 Synthetic 
                 Integrated DNA  
               
               
                   
                   
                   
                 Technologies, Inc. 
               
               
                 MBIS 
                 
                   S. cerevisiae 
                 
                 pMBIS 
                 Addgene: 17817 
               
               
                 ADS 
                 
                   Artemisia  
                 
                 pADS 
                 Addgene: 19040 
               
               
                   
                 
                   annua 
                 
                   
                   
               
               
                 GHS 
                 
                   Abies grandis 
                 
                 pTrcHUM 
                 Addgene: 19003 
               
               
                 ABS 
                 
                   Abies grandis 
                 
                 pSBET/ 
                 Ruben Peters, Iowa  
               
               
                   
                   
                 AgAs 
                 State University 
               
               
                 TXS 
                 
                   Taxus  
                 
                 M60 
                 David W.  
               
               
                   
                 
                   brevifola 
                 
                   
                 Christianson, University  
               
               
                   
                   
                   
                 of Pennsylvania 
               
               
                 GGPPS 
                 
                   Taxus  
                 
                 gBlock 
                 Integrated DNA  
               
               
                   
                 
                   Canadensis 
                 
                   
                 Technologies, Inc. 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Plasmids 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Anti- 
                 Add- 
               
               
                 Plasmid 
                 Description 
                 biotic* 
                 gene 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 F-plasmid 
                 The F-plasmid from the  
                 T 
                 105063 
               
               
                   
                 S1030 strain of  E. coli.   
                   
                   
               
               
                 pB2H 1b   
                 An early version of B2H that  
                 K 
                 TBD 
               
               
                   
                 lacks PTP1B and contains 
                   
                   
               
               
                   
                 LuxAB as the GOT 
                   
                   
               
               
                 pBAD 1b.Src   
                 Enables inducible expression  
                 P 
                 TBD 
               
               
                   
                 of Src and CDC37 
                   
                   
               
               
                 pBAD 1b.SH2   
                 Enables inducible expression  
                 P 
                 TBD 
               
               
                   
                 of the SH2 domain. 
                   
                   
               
               
                 pBAD 1b.S   
                 Enables inducible expression  
                 P 
                 TBD 
               
               
                   
                 of the substrate domain. 
                   
                   
               
               
                 pBAD 1b.All   
                 Enables inducible expression  
                 P 
                 TBD 
               
               
                   
                 of Src, CDC37, the SH2 
                   
                   
               
               
                   
                 domain, and the substrate domain. 
                   
                   
               
               
                 pB2H 1c.p130cas   
                 An early version of B2H that (i)  
                 K 
                 TBD 
               
               
                   
                 lacks PTP1B and Src, (ii) 
                   
                   
               
               
                   
                 contains LuxAB, and (iii)  
                   
                   
               
               
                   
                 includes a substrate from 
                   
                   
               
               
                   
                 p130cas. 
                   
                   
               
               
                 pB2H 1c.midT   
                 An early version of B2H that (i)  
                 K 
                 TBD 
               
               
                   
                 lacks PTP1B and Src, (ii) 
                   
                   
               
               
                   
                 contains LuxAB, and (iii)  
                   
                   
               
               
                   
                 includes a substrate from midT. 
                   
                   
               
               
                 pB2H 1c.ShcA   
                 An early version of B2H that (i)  
                 K 
                 TBD 
               
               
                   
                 lacks PTP1B and Src, (ii) 
                   
                   
               
               
                   
                 contains LuxAB, and (iii) includes  
                   
                   
               
               
                   
                 a substrate from ShCA. 
                   
                   
               
               
                 pB2H 1c.EGFR   
                 An early version of B2H that (i)  
                 K 
                 TBD 
               
               
                   
                 lacks PTP1B and Src, (ii) 
                   
                   
               
               
                   
                 contains LuxAB, and (iii) includes  
                   
                   
               
               
                   
                 a substrate from EGFR. 
                   
                   
               
               
                 pBAD 1c   
                 Enables inducible expression  
                 P 
                 TBD 
               
               
                   
                 of Src and CDC37. 
                   
                   
               
               
                 pBAD 1d   
                 Enables inducible expression  
                 P 
                 TBD 
               
               
                   
                 of Src and PTP1B. 
                   
                   
               
               
                 pBAD 1d.mut   
                 Enables inducible expression  
                 P 
                 TBD 
               
               
                   
                 of Src and catalytically 
                   
                   
               
               
                   
                 inactive PTP1B (C215S). 
                   
                   
               
               
                 pB2H S1.1Pro1   
                 An early version of B2H that (i)  
                 K 
                 TBD 
               
               
                   
                 lacks PTP1B, (ii) contains 
                   
                   
               
               
                   
                 LuxAB, (iii) places expression  
                   
                   
               
               
                   
                 of Src, CDC37, the SH2 
                   
                   
               
               
                   
                 domain, and the substrate  
                   
                   
               
               
                   
                 domain under control of the 
                   
                   
               
               
                   
                 same Pro1 promoter, and (iv)  
                   
                   
               
               
                   
                 uses the BB034 RBS for Src. 
                   
                   
               
               
                 pB2H S1.1Pro1.   
                 Identical to pB2H S1.1Pro1   
                 K 
                 TBD 
               
               
                 
                   mut 
                 
                 except for a mutation in the 
                   
                   
               
               
                   
                 substrate (Y4F) 
                   
                   
               
               
                 pB2H S1.1ProD   
                 An early version of B2H that (i)  
                 K 
                 TBD 
               
               
                   
                 lacks PTP1B, (ii) contains 
                   
                   
               
               
                   
                 LuxAB, and (iii) includes the ProD  
                   
                   
               
               
                   
                 promoter and pro RBS for Src. 
                   
                   
               
               
                 pB2H S1.1ProD.   
                 Identical to pB2H S1.1ProD   
                 K 
                 TBD 
               
               
                 
                   mut 
                 
                 except for a mutation in the 
                   
                   
               
               
                   
                 substrate (Y4F) 
                   
                   
               
               
                 pB2H S1.2pro   
                 An early version of B2H that  
                 K 
                 TBD 
               
               
                   
                 (i) lacks PTP1B, (ii) contains 
                   
                   
               
               
                   
                 LuxAB, and (iii) includes  
                   
                   
               
               
                   
                 the pro RBS for Src. 
                   
                   
               
               
                 pB2H S1.2pro.   
                 Identical to pB2H S1.2pro   
                 K 
                 TBD 
               
               
                 
                   mut 
                 
                 except for a mutation in the 
                   
                   
               
               
                   
                 substrate (Y4F) 
                   
                   
               
               
                 pB2H S1.2Sal28   
                 An early version of B2H that (i)  
                 K 
                 TBD 
               
               
                   
                 lacks PTP1B, (ii) contains 
                   
                   
               
               
                   
                 LuxAB, and (iii) includes  
                   
                   
               
               
                   
                 the Sal28 RBS for Src. 
                   
                   
               
               
                 pB2H S1.2Sal28.   
                 Identical to pB2H S1.2Sal28  except  
                 K 
                 TBD 
               
               
                 
                   mut 
                 
                 for a mutation in the substrate (Y4F) 
                   
                   
               
               
                 pB2H S1.3RBS30   
                 An early version of B2H that  
                 K 
                 TBD 
               
               
                   
                 (i) contains LuxAB and (ii) 
                   
                   
               
               
                   
                 includes the bb030 RBS for PTP1B. 
                   
                   
               
               
                 pB2H S1.3RBS30   
                 Identical to pB2H S1.3RBS30    
                 K 
                 TBD 
               
               
                   
                 except for a mutation in the 
                   
                   
               
               
                   
                 substrate (Y4F) 
                   
                   
               
               
                 pB2H S1.3RBS34   
                 An early version of B2H that  
                 K 
                 TBD 
               
               
                   
                 (i) contains LuxAB and (ii) 
                   
                   
               
               
                   
                 includes the bb034 RBS for PTP1B. 
                   
                   
               
               
                 pB2H S1.3RBS34   
                 Identical to pB2H S1.3RBS34   
                 K 
                 TBD 
               
               
                   
                 except for a mutation in the 
                   
                   
               
               
                   
                 substrate (Y4F) 
                   
                   
               
               
                 pB2H S2RBS30   
                 An early version of B2H that  
                 K 
                 TBD 
               
               
                   
                 (i) contains SpecR and (ii) 
                   
                   
               
               
                   
                 includes the bb030 RBS for PTP1B. 
                   
                   
               
               
                 pB2H S2RBS30.   
                 Identical to pB2H S2RBS30   
                 K 
                 TBD 
               
               
                 
                   mut 
                 
                 except for an inactivating 
                   
                   
               
               
                   
                 mutation in PTP1B (C215S) 
                   
                   
               
               
                 pB2H opt   
                 Final, optimized B2H that  
                 K 
                 TBD 
               
               
                   
                 (i) contains SpecR and (ii) 
                   
                   
               
               
                   
                 includes the bb034 RBS for PTP1B. 
                   
                   
               
               
                 pB2H opt * 
                 Identical to pB2H opt  except for  
                 K 
                 TBD 
               
               
                   
                 an inactivating mutation in 
                   
                   
               
               
                   
                 PTP1B (C215S) 
                   
                   
               
               
                 pB2H optX   
                   
                 K 
                 TBD 
               
               
                 pMBIS 
                 A plasmid that harbors genes  
                 T 
                 17817 
               
               
                   
                 for the mevalonate- 
                   
                   
               
               
                   
                 dependent isoprenoid pathway  
                   
                   
               
               
                   
                 from  S. cerevisiae  and 
                   
                   
               
               
                   
                 harbors a tetracycline  
                   
                   
               
               
                   
                 resistance marker. 
                   
                   
               
               
                 pMBIS CmR   
                 A plasmid that harbors  
                 P 
                 TBD 
               
               
                   
                 genes for the mevalonate- 
                   
                   
               
               
                   
                 dependent isoprenoid pathway  
                   
                   
               
               
                   
                 from  S. cerevisiae  and 
                   
                   
               
               
                   
                 harbors a chloramphenicol  
                   
                   
               
               
                   
                 resistance marker. 
                   
                   
               
               
                 pTrc99t 
                 A pTrc99a variant with BsaI 
                 C 
                 TBD 
               
               
                   
                 removed for use in Golden 
                   
                   
               
               
                   
                 Gate cloning 
                   
                   
               
               
                 PTS ADS   
                 A plasmid that harbors ADS. 
                 C 
                 TBD 
               
               
                 PTS ADS(G349A)   
                 A plasmid that harbors ADS (G349A). 
                 C 
                 TBD 
               
               
                 PTS ADS(G400C)   
                 A plasmid that harbors ADS (G400C). 
                 C 
                 TBD 
               
               
                 PTS ADS(D299A)   
                 A plasmid that harbors ADS  
                 C 
                 TBD 
               
               
                   
                 (D299A, inactivating). 
                   
                   
               
               
                 PTS ADS(F514E)   
                 A plasmid that harbors ADS (F514E). 
                 C 
                 TBD 
               
               
                 pTS ADS(G400L)   
                 A plasmid that harbors ADS (G400L). 
                 C 
                 TBD 
               
               
                 PTS ADS(F514S)   
                 A plasmid that harbors ADS (F514S). 
                 C 
                 TBD 
               
               
                 PTS ADS(F514V)   
                 A plasmid that harbors ADS (F514V). 
                 C 
                 TBD 
               
               
                 pTS ADS(V292I)   
                 A plasmid that harbors ADS (V292I). 
                 C 
                 TBD 
               
               
                 PTS ADS(I90S/   
                 A plasmid that harbors ADS  
                 C 
                 TBD 
               
               
                 
                   F340S) 
                 
                 (I90S/F340S). 
                   
                   
               
               
                 PTS ADS(I490V/   
                 A plasmid that harbors ADS  
                 C 
                 TBD 
               
               
                 
                   M528K) 
                 
                 (I490V/M528K). 
                   
                   
               
               
                 PTS ADS(G34S/   
                 A plasmid that harbors ADS  
                 C 
                 TBD 
               
               
                 
                   K51N) 
                 
                 (G34S/K51N). 
                   
                   
               
               
                 pTS ADSF370Y   
                 A plasmid that harbors ADS (F370Y). 
                 C 
                 TBD 
               
               
                 PTS ADSR527L   
                 A plasmid that harbors ADS (R527L). 
                 C 
                 TBD 
               
               
                 pTS GHS   
                 A plasmid that harbors GHS. 
                 C 
                 TBD 
               
               
                 pTS GHS(BFN)   
                 A plasmid that harbors GHS  
                 C 
                 TBD 
               
               
                   
                 (W315P). 
                   
                   
               
               
                 PTS GHS(SIB)   
                 A plasmid that harbors GHS  
                 C 
                 TBD 
               
               
                   
                 (F312Q/M339A/M447F). 
                   
                   
               
               
                 PTS GHS(HUM)   
                 A plasmid that harbors GHS  
                 C 
                 TBD 
               
               
                   
                 (M339N/S484C/M565I). 
                   
                   
               
               
                 pTS GHS(BBA)   
                 A plasmid that harbors GHS  
                 C 
                 TBD 
               
               
                   
                 (A336V/M447H/I562T). 
                   
                   
               
               
                 pTS GHS(ALP)   
                 A plasmid that harbors GHS 
                 C 
                 TBD 
               
               
                   
                 (A336C/T445C/S484C/ 
                   
                   
               
               
                   
                 I562L/M565L). 
                   
                   
               
               
                 pTS GHS(LFN)   
                 A plasmid that harbors GHS 
                 C 
                 TBD 
               
               
                   
                 (A317N/A337S/S484C/I562V). 
                   
                   
               
               
                 pTS GHS(A319Q)   
                 A plasmid that harbors GHS  
                 C 
                 TBD 
               
               
                   
                 (A319Q). 
                   
                   
               
               
                 pTSGHS 
                 A plasmid that harbors GHS (S561C). 
                 C 
                 TBD 
               
               
                 (S561C) 
                   
                   
                   
               
               
                 pTSGHS 
                 A plasmid that harbors GHS  
                 C 
                 TBD 
               
               
                 (Y415C) 
                 (Y415C). 
                   
                   
               
               
                 pTS GHS(S484L)   
                 A plasmid that harbors GHS (S484L). 
                 C 
                 TBD 
               
               
                 PTS GHS(450Y)   
                 A plasmid that harbors GHS (L450Y). 
                 C 
                 TBD 
               
               
                 PTS GHS(450G)   
                 A plasmid that harbors GHS (L450G). 
                 C 
                 TBD 
               
               
                 PTS GHS(450K)   
                 A plasmid that harbors GHS (L450K). 
                 C 
                 TBD 
               
               
                 PTS GHS(450T)   
                 A plasmid that harbors GHS (L450T). 
                 C 
                 TBD 
               
               
                 pTS GHS(T455I)   
                 A plasmid that harbors GHS (T455I). 
                 C 
                 TBD 
               
               
                 PTS ABS   
                 A plasmid that harbors  
                 C 
                 TBD 
               
               
                   
                 ABS and GGPPS. 
                   
                   
               
               
                 PTS TXS   
                 A plasmid that harbors  
                 C 
                 TBD 
               
               
                   
                 TXS and GGPPS. 
               
               
                   
               
               
                 *Antibiotic resistance: carbenicillin (C, 50 μg/ml), kanamycin (K, 50 μg/ml), tetracycline (T, 10 μg/ml), chloramphenicol (P, 34 μg/ml), and spectinomycin (S, conditional). 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Components of various B2H systems. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 DNA 
                   
                 Amino Acid 
                   
               
               
                   
                   
                 SEQ 
                   
                 SEQ 
                   
               
               
                 Component 
                 Name 
                 ID NO: 
                 DNA 
                 ID NO: 
                 Amino Acid 
               
               
                   
               
               
                 Kinase 
                 c-Src 
                  3 
                 ATGGGCTCCAAGCCGCAGACTCAGG 
                 21 
                 MGSKPQTQGLAKDAWEIP 
               
               
                   
                   
                   
                 GCCTGGCCAAGGATGCCTGGGAGAT 
                   
                 RESLRLEVKLGQGCFGEV 
               
               
                   
                   
                   
                 CCCTCGGGAGTCGCTGCGGCTGGAG 
                   
                 WMGTWNGTTRVAIKTLKP 
               
               
                   
                   
                   
                 GTCAAGCTGGGCCAGGGCTGCTTTG 
                   
                 GTMSPEAFLQEAQVMKKL 
               
               
                   
                   
                   
                 GCGAGGTGTGGATGGGGACCTGGAA 
                   
                 RHEKLVQLYAVVSEEPIYIV 
               
               
                   
                   
                   
                 CGGTACCACCAGGGTGGCCATCAAA 
                   
                 TEYMSKGSLLDFLKGETGK 
               
               
                   
                   
                   
                 ACCCTGAAGCCTGGCACGATGTCTC 
                   
                 YLRLPQLVDMAAQIASGM 
               
               
                   
                   
                   
                 CAGAGGCCTTCCTGCAGGAGGCCCA 
                   
                 AYVERMNYVHRDLRAANI 
               
               
                   
                   
                   
                 GGTCATGAAGAAGCTGAGGCATGAG 
                   
                 LVGENLVCKVADFGLARLI 
               
               
                   
                   
                   
                 AAGCTGGTGCAGTTGTATGCTGTGG 
                   
                 EDNEYTARQGAKFPIKWTA 
               
               
                   
                   
                   
                 TTTCAGAGGAGCCCATTTACATCGT 
                   
                 PEAALYGRFTIKSDVWSFGI 
               
               
                   
                   
                   
                 CACGGAGTACATGAGCAAGGGGAG 
                   
                 LLTELTTKGRVPYPGMVNR 
               
               
                   
                   
                   
                 TTTGCTGGACTTTCTCAAGGGGGAG 
                   
                 EVLDQVERGYRMPCPPECP 
               
               
                   
                   
                   
                 ACAGGCAAGTACCTGCGGCTGCCTC 
                   
                 ESLHDLMCQCWRKEPEERP 
               
               
                   
                   
                   
                 AGCTGGTGGACATGGCTGCTCAGAT 
                   
                 TFEYLQAFLEDYFTSTEPQY 
               
               
                   
                   
                   
                 CGCCTCAGGCATGGCGTACGTGGAG 
                   
                 QPGENL* 
               
               
                   
                   
                   
                 CGGATGAACTACGTCCACCGGGACC 
                   
                   
               
               
                   
                   
                   
                 TTCGTGCAGCCAACATCCTGGTGGG 
                   
                   
               
               
                   
                   
                   
                 AGAGAACCTGGTGTGCAAAGTGGCC 
                   
                   
               
               
                   
                   
                   
                 GACTTTGGGCTGGCTCGGCTCATTG 
                   
                   
               
               
                   
                   
                   
                 AAGACAATGAGTACACGGCGCGGC 
                   
                   
               
               
                   
                   
                   
                 AAGGTGCCAAATTCCCCATCAAGTG 
                   
                   
               
               
                   
                   
                   
                 GACGGCTCCAGAAGCTGCCCTCTAT 
                   
                   
               
               
                   
                   
                   
                 GGCCGCTTCACCATCAAGTCGGACG 
                   
                   
               
               
                   
                   
                   
                 TGTGGTCCTTCGGGATCCTGCTGACT 
                   
                   
               
               
                   
                   
                   
                 GAGCTCACCACAAAGGGACGGGTGC 
                   
                   
               
               
                   
                   
                   
                 CCTACCCTGGGATGGTGAACCGCGA 
                   
                   
               
               
                   
                   
                   
                 GGTGCTGGACCAGGTGGAGCGGGGC 
                   
                   
               
               
                   
                   
                   
                 TACCGGATGCCCTGCCCGCCGGAGT 
                   
                   
               
               
                   
                   
                   
                 GTCCCGAGTCCCTGCACGACCTCAT 
                   
                   
               
               
                   
                   
                   
                 GTGCCAGTGCTGGCGGAAGGAGCCT 
                   
                   
               
               
                   
                   
                   
                 GAGGAGCGGCCCACCTTCGAGTACC 
                   
                   
               
               
                   
                   
                   
                 TGCAGGCCTTCCTGGAGGACTACTT 
                   
                   
               
               
                   
                   
                   
                 CACGTCCACCGAGCCCCAGTACCAG 
                   
                   
               
               
                   
                   
                   
                 CCCGGGGAGAACCTCTAA 
                   
                   
               
               
                   
               
               
                 Chaperone 
                 CDC37 
                  4 
                 ATGGTGGACTACAGCGTGTGGGACC 
                 22 
                 MVDYSVWDHIEVSDDEDE 
               
               
                   
                   
                   
                 ACATTGAGGTGTCTGATGATGAAGA 
                   
                 THPNIDTASLFRWRHQARV 
               
               
                   
                   
                   
                 CGAGACGCACCCCAACATCGACACG 
                   
                 ERMEQFQKEKEELDRGCRE 
               
               
                   
                   
                   
                 GCCAGTCTCTTCCGCTGGCGGCATC 
                   
                 CKRKVAECQRKLKELEVA 
               
               
                   
                   
                   
                 AGGCCCGGGTGGAACGCATGGAGC 
                   
                 EGGKAELERLQAEAQQLR 
               
               
                   
                   
                   
                 AGTTCCAGAAGGAGAAGGAGGAAC 
                   
                 KEERSWEQKLEEMRKKEK 
               
               
                   
                   
                   
                 TGGACAGGGGCTGCCGCGAGTGCAA 
                   
                 SMPWNVDTLSKDGFSKSM 
               
               
                   
                   
                   
                 GCGCAAGGTGGCCGAGTGCCAGAG 
                   
                 VNTKPEKTEEDSEEVREQK 
               
               
                   
                   
                   
                 GAAACTGAAGGAGCTGGAGGTGGC 
                   
                 HKTFVEKYEKQIKHFGMLR 
               
               
                   
                   
                   
                 CGAGGGCGGCAAGGCAGAGCTGGA 
                   
                 RWDDSQKYLSDNVHLVCE 
               
               
                   
                   
                   
                 GCGCCTGCAGGCCGAGGCACAGCAG 
                   
                 ETANYLVIWCIDLEVEEKC 
               
               
                   
                   
                   
                 CTGCGCAAGGAGGAGCGGAGCTGG 
                   
                 ALMEQVAHQTIVMQFILEL 
               
               
                   
                   
                   
                 GAGCAGAAGCTGGAGGAGATGCGC 
                   
                 AKSLKVDPRACFRQFFTKI 
               
               
                   
                   
                   
                 AAGAAGGAGAAGAGCATGCCCTGG 
                   
                 KTADRQYMEGFNDELEAF 
               
               
                   
                   
                   
                 AACGTGGACACGCTCAGCAAAGACG 
                   
                 KERVRGRAKLRIEKAMKE 
               
               
                   
                   
                   
                 GCTTCAGCAAGAGCATGGTAAATAC 
                   
                 YEEEERKKRLGPGGLDPVE 
               
               
                   
                   
                   
                 CAAGCCCGAGAAGACGGAGGAGGA 
                   
                 VYESLPEELQKCFDVKDVQ 
               
               
                   
                   
                   
                 CTCAGAGGAGGTGAGGGAGCAGAA 
                   
                 MLQDAISKMDPTDAKYHM 
               
               
                   
                   
                   
                 ACACAAGACCTTCGTGGAAAAATAC 
                   
                 QRCIDSGLWVPNSKASEAK 
               
               
                   
                   
                   
                 GAGAAACAGATCAAGCACTTTGGCA 
                   
                 EGEEAGPGDPLLEAVPKTG 
               
               
                   
                   
                   
                 TGCTTCGCCGCTGGGATGACAGCCA 
                   
                 DEKDVSV* 
               
               
                   
                   
                   
                 AAAGTACCTGTCAGACAACGTCCAC 
                   
                   
               
               
                   
                   
                   
                 CTGGTGTGCGAGGAGACAGCCAATT 
                   
                   
               
               
                   
                   
                   
                 ACCTGGTCATTTGGTGCATTGACCTA 
                   
                   
               
               
                   
                   
                   
                 GAGGTGGAGGAGAAATGTGCACTCA 
                   
                   
               
               
                   
                   
                   
                 TGGAGCAGGTGGCCCACCAGACAAT 
                   
                   
               
               
                   
                   
                   
                 CGTCATGCAATTTATCCTGGAGCTG 
                   
                   
               
               
                   
                   
                   
                 GCCAAGAGCCTAAAGGTGGACCCCC 
                   
                   
               
               
                   
                   
                   
                 GGGCCTGCTTCCGGCAGTTCTTCACT 
                   
                   
               
               
                   
                   
                   
                 AAGATTAAGACAGCCGATCGCCAGT 
                   
                   
               
               
                   
                   
                   
                 ACATGGAGGGCTTCAACGACGAGCT 
                   
                   
               
               
                   
                   
                   
                 GGAAGCCTTCAAGGAGCGTGTGCGG 
                   
                   
               
               
                   
                   
                   
                 GGCCGTGCCAAGCTGCGCATCGAGA 
                   
                   
               
               
                   
                   
                   
                 AGGCCATGAAGGAGTACGAGGAGG 
                   
                   
               
               
                   
                   
                   
                 AGGAGCGCAAGAAGCGGCTCGGCC 
                   
                   
               
               
                   
                   
                   
                 CCGGCGGCCTGGACCCCGTCGAGGT 
                   
                   
               
               
                   
                   
                   
                 CTACGAGTCCCTCCCTGAGGAACTC 
                   
                   
               
               
                   
                   
                   
                 CAGAAGTGCTTCGATGTGAAGGACG 
                   
                   
               
               
                   
                   
                   
                 TGCAGATGCTGCAGGACGCCATCAG 
                   
                   
               
               
                   
                   
                   
                 CAAGATGGACCCCACCGACGCAAAG 
                   
                   
               
               
                   
                   
                   
                 TACCACATGCAGCGCTGCATTGACT 
                   
                   
               
               
                   
                   
                   
                 CTGGCCTCTGGGTCCCCAACTCTAA 
                   
                   
               
               
                   
                   
                   
                 GGCCAGCGAGGCCAAGGAGGGAGA 
                   
                   
               
               
                   
                   
                   
                 GGAGGCAGGTCCTGGGGACCCATTA 
                   
                   
               
               
                   
                   
                   
                 CTGGAAGCTGTTCCCAAGACGGGCG 
                   
                   
               
               
                   
                   
                   
                 ATGAGAAGGATGTCAGTGTGTAA 
                   
                   
               
               
                   
               
               
                 Phosphatase 
                 PTP1B 
                  5 
                 ATGGAGATGGAAAAGGAGTTCGAG 
                 23 
                 MEMEKEFEQIDKSGSWAAI 
               
               
                   
                   
                   
                 CAGATCGACAAGTCCGGGAGCTGGG 
                   
                 YQDIRHEASDFPCRVAKLP 
               
               
                   
                   
                   
                 CGGCCATTTACCAGGATATCCGACA 
                   
                 KNKNRNRYRDVSPFDHSRI 
               
               
                   
                   
                   
                 TGAAGCCAGTGACTTCCCATGTAGA 
                   
                 KLHQEDNDYINASLIKMEE 
               
               
                   
                   
                   
                 GTGGCCAAGCTTCCTAAGAACAAAA 
                   
                 AQRSYILTQGPLPNTCGHF 
               
               
                   
                   
                   
                 ACCGAAATAGGTACAGAGACGTCAG 
                   
                 WEMVWEQKSRGVVMLNR 
               
               
                   
                   
                   
                 TCCCTTTGACCATAGTCGGATTAAA 
                   
                 VMEKGSLKCAQYWPQKEE 
               
               
                   
                   
                   
                 CTACATCAAGAAGATAATGACTATA 
                   
                 KEMIFEDTNLKLTLISEDIK 
               
               
                   
                   
                   
                 TCAACGCTAGTTTGATAAAAATGGA 
                   
                 SYYTVRQLELENLTTQETR 
               
               
                   
                   
                   
                 AGAAGCCCAAAGGAGTTACATTCTT 
                   
                 EILHFHYTTWPDFGVPESPA 
               
               
                   
                   
                   
                 ACCCAGGGCCCTTTGCCTAACACAT 
                   
                 SFLNFLFKVRESGSLSPEHG 
               
               
                   
                   
                   
                 GCGGTCACTTTTGGGAGATGGTGTG 
                   
                 PVVVHCSAGIGRSGTFCLA 
               
               
                   
                   
                   
                 GGAGCAGAAAAGCAGGGGTGTCGT 
                   
                 DTCLLLMDKRKDPSSVDIK 
               
               
                   
                   
                   
                 CATGCTCAACAGAGTGATGGAGAAA 
                   
                 KVLLEMRKFRMGLIQTAD 
               
               
                   
                   
                   
                 GGTTCGTTAAAATGCGCACAATACT 
                   
                 QLRFSYLAVIEGAKFIMGD 
               
               
                   
                   
                   
                 GGCCACAAAAAGAAGAAAAAGAGA 
                   
                 SSVQDQWKELSHEDLEPPP 
               
               
                   
                   
                   
                 TGATCTTTGAAGACACAAATTTGAA 
                   
                 EHIPPPPRPPKRILEPHN* 
               
               
                   
                   
                   
                 ATTAACATTGATCTCTGAAGATATC 
                   
                   
               
               
                   
                   
                   
                 AAGTCATATTATACAGTGCGACAGC 
                   
                   
               
               
                   
                   
                   
                 TAGAATTGGAAAACCTTACAACCCA 
                   
                   
               
               
                   
                   
                   
                 AGAAACTCGAGAGATCTTACATTTC 
                   
                   
               
               
                   
                   
                   
                 CACTATACCACATGGCCTGACTTTG 
                   
                   
               
               
                   
                   
                   
                 GAGTCCCTGAATCACCAGCCTCATT 
                   
                   
               
               
                   
                   
                   
                 CTTGAACTTTCTTTTCAAAGTCCGAG 
                   
                   
               
               
                   
                   
                   
                 AGTCAGGGTCACTCAGCCCGGAGCA 
                   
                   
               
               
                   
                   
                   
                 CGGGCCCGTTGTGGTGCACTGCAGT 
                   
                   
               
               
                   
                   
                   
                 GCAGGCATCGGCAGGTCTGGAACCT 
                   
                   
               
               
                   
                   
                   
                 TCTGTCTGGCTGATACCTGCCTCTTG 
                   
                   
               
               
                   
                   
                   
                 CTGATGGACAAGAGGAAAGACCCTT 
                   
                   
               
               
                   
                   
                   
                 CTTCCGTTGATATCAAGAAAGTGCT 
                   
                   
               
               
                   
                   
                   
                 GTTAGAAATGAGGAAGTTTCGGATG 
                   
                   
               
               
                   
                   
                   
                 GGGCTGATCCAGACAGCCGACCAGC 
                   
                   
               
               
                   
                   
                   
                 TGCGCTTCTCCTACCTGGCTGTGATC 
                   
                   
               
               
                   
                   
                   
                 GAAGGTGCCAAATTCATCATGGGGG 
                   
                   
               
               
                   
                   
                   
                 ACTCTTCCGTGCAGGATCAGTGGAA 
                   
                   
               
               
                   
                   
                   
                 GGAGCTTTCCCACGAGGACCTGGAG 
                   
                   
               
               
                   
                   
                   
                 CCCCCACCCGAGCATATCCCCCCAC 
                   
                   
               
               
                   
                   
                   
                 CTCCCCGGCCACCCAAACGAATCCT 
                   
                   
               
               
                   
                   
                   
                 GGAGCCACACAATTGA 
                   
                   
               
               
                   
               
               
                 Substrate 
                 p130cas 
                  6 
                 TGGATGGAGGACTATGACTACGTCC 
                 24 
                 WMEDYDYVHLQG 
               
               
                   
                   
                   
                 ACCTACAGGGG 
                   
                   
               
               
                   
               
               
                 Substrate 
                 midT 
                  7 
                 GAACCGCAGTATGAAGAAATTCCGA 
                 25 
                 EPQYEEIPIYL 
               
               
                   
                   
                   
                 TTTATCTG 
                   
                   
               
               
                   
               
               
                 Substrate 
                 ShcA 
                  8 
                 GATCATCAGTATTATAACGATTTTCC 
                 26 
                 DHQYYNDFPG 
               
               
                   
                   
                   
                 GGGC 
                   
                   
               
               
                   
               
               
                 Substrate 
                 EGFR 
                  9 
                 CCGCAGCGCTATCTGGTGATTCAGG 
                 27 
                 PQRYLVIQGD 
               
               
                   
                   
                   
                 GCGAT 
                   
                   
               
               
                   
               
               
                 Substrate 
                 p130cas 
                 10 
                 TGGATGGAGGACTTTGACTTCGTCC 
                 28 
                 WMEDFDFVHLQG 
               
               
                   
                 Y/F 
                   
                 ACCTACAGGGG 
                   
                   
               
               
                   
               
               
                 Substrate 
                 midT Y/F 
                 11 
                 GAACCGCAGTTTGAAGAAATTCCGA 
                 29 
                 EPQFEEIPIYL 
               
               
                   
                   
                   
                 TTTATCTG 
                   
                   
               
               
                   
               
               
                 Promoter 
                 pBAD 
                 12 
                 AGAAACCAATTGTCCATATTGCATC 
                 — 
                 N/A 
               
               
                   
                   
                   
                 AGACATTGCCGTCACTGCGTCTTTTA 
                   
                   
               
               
                   
                   
                   
                 CTGGCTCTTCTCGCTAACCAAACCG 
                   
                   
               
               
                   
                   
                   
                 GTAACCCCGCTTATTAAAAGCATTC 
                   
                   
               
               
                   
                   
                   
                 TGTAACAAAGCGGGACCAAAGCCAT 
                   
                   
               
               
                   
                   
                   
                 GACAAAAACGCGTAACAAAAGTGTC 
                   
                   
               
               
                   
                   
                   
                 TATAATCACGGCAGAAAAGTCCACA 
                   
                   
               
               
                   
                   
                   
                 TTGATTATTTGCACGGCGTCACACTT 
                   
                   
               
               
                   
                   
                   
                 TGCTATGCCATAGCATTTTTATCCAT 
                   
                   
               
               
                   
                   
                   
                 AAGATTAGCG 
                   
                   
               
               
                   
               
               
                 Promoter 
                 Pro1 57   
                 13 
                 TTCTAGAGCACAGCTAACACCACGT 
                   
                 N/A 
               
               
                   
                   
                   
                 CGTCCCTATCTGCTGCCCTAGGTCTA 
                   
                   
               
               
                   
                   
                   
                 TGAGTGGTTGCTGGATAACTTTACG 
                   
                   
               
               
                   
                   
                   
                 GGCATGCATAAGGCTCGGTATCTAT 
                   
                   
               
               
                   
                   
                   
                 ATTCAGGGAGACCACAACGGTTTCC 
                   
                   
               
               
                   
                   
                   
                 CTCTACAAATAATTTTGTTTAACTTT 
                   
                   
               
               
                   
                   
                   
                 TACTAGAG 
                   
                   
               
               
                   
               
               
                 Promoter 
                 placZopt 39   
                 14 
                 CATTAGGCACCCCGGGCTTTACTCG 
                   
                 N/A 
               
               
                   
                   
                   
                 TAAAGCTTCCGGCGCGTATGTTGTG 
                   
                   
               
               
                   
                   
                   
                 TCGACCG 
                   
                   
               
               
                   
               
               
                 Promoter 
                 ProD 57   
                 13 
                 TTCTAGAGCACAGCTAACACCACGT 
                   
                 N/A 
               
               
                   
                   
                   
                 CGTCCCTATCTGCTGCCCTAGGTCTA 
                   
                   
               
               
                   
                   
                   
                 TGAGTGGTTGCTGGATAACTTTACG 
                   
                   
               
               
                   
                   
                   
                 GGCATGCATAAGGCTCGGTATCTAT 
                   
                   
               
               
                   
                   
                   
                 ATTCAGGGAGACCACAACGGTTTCC 
                   
                   
               
               
                   
                   
                   
                 CTCTACAAATAATTTTGTTTAACTTT 
                   
                   
               
               
                   
                   
                   
                 TACTAGAG 
                   
                   
               
               
                   
               
               
                 RBS 
                 Pro 
                 15 
                 GTGCAGTTAAAGAGGAGAAAGGTC 
                   
                 N/A 
               
               
                   
               
               
                 RBS 
                 Sal28 ‡   
                 16 
                 CGAAAAAAAGTAAGGCGGTAATCC 
                   
                 N/A 
               
               
                   
               
               
                 RBS 
                 BB030 
                 17 
                 TCTAGAGATTAAAGAGGAGAAATAC 
                   
                 N/A 
               
               
                   
                   
                   
                 TAG 
                   
                   
               
               
                   
               
               
                 RBS 
                 BB034 
                 18 
                 TCTAGAAAAGAGGAGAAATACTAG 
                   
                 N/A 
               
               
                   
               
               
                 GOI 
                 LuxAB 
                 19 
                 ATGAAATTTGGAAACTTTTTGCTTAC 
                 30 
                 MKFGNFLLTYQPPQFSQTE 
               
               
                   
                   
                   
                 ATACCAACCTCCCCAATTTTCCCAA 
                   
                 VMKRLVKLGRISEECGFDT 
               
               
                   
                   
                   
                 ACAGAGGTAATGAAACGTTTGGTTA 
                   
                 VWLLEHHFTEFGLLGNPYV 
               
               
                   
                   
                   
                 AATTAGGTCGCATCTCTGAGGAGTG 
                   
                 AAAYLLGATKKLNVGTAA 
               
               
                   
                   
                   
                 TGGTTTTGATACCGTATGGTTACTGG 
                   
                 IVLPTAHPVRQLEDVNLLD 
               
               
                   
                   
                   
                 AGCATCATTTCACGGAGTTTGGTTTG 
                   
                 QMSKGRFRFGICRGLYNKD 
               
               
                   
                   
                   
                 CTTGGTAACCCTTATGTCGCTGCTGC 
                   
                 FRVFGTDMNNSRALAECW 
               
               
                   
                   
                   
                 ATATTTACTTGGCGCGACTAAAAAA 
                   
                 YGLIKNGMTEGYMEADNE 
               
               
                   
                   
                   
                 TTGAATGTAGGAACTGCCGCTATTG 
                   
                 HIKFHKVKVNPAAYSRGG 
               
               
                   
                   
                   
                 TTCTTCCCACAGCCCATCCAGTACGC 
                   
                 APVYVVAESASTTEWAAQ 
               
               
                   
                   
                   
                 CAACTTGAAGATGTGAATTTATTGG 
                   
                 FGLPMILSWIINTNEKKAQL 
               
               
                   
                   
                   
                 ATCAAATGTCAAAAGGACGATTTCG 
                   
                 ELYNEVAQEYGHDIHNIDH 
               
               
                   
                   
                   
                 GTTTGGTATTTGCCGAGGGCTTTACA 
                   
                 CLSYITSVDHDSIKAKEICR 
               
               
                   
                   
                   
                 ACAAGGACTTTCGCGTATTCGGCAC 
                   
                 KFLGHWYDSYVNATTIFDD 
               
               
                   
                   
                   
                 AGATATGAATAACAGTCGCGCCTTA 
                   
                 SDQTRGYDFNKGQWRDFV 
               
               
                   
                   
                   
                 GCGGAATGCTGGTACGGGCTGATAA 
                   
                 LKGHKDTNRRIDYSYEINP 
               
               
                   
                   
                   
                 AGAATGGCATGACAGAGGGATATAT 
                   
                 VGTPQECIDIIQKDIDATGIS 
               
               
                   
                   
                   
                 GGAAGCTGATAATGAACATATCAAG 
                   
                 NICCGFEANGTVDEIIASMK 
               
               
                   
                   
                   
                 TTCCATAAGGTAAAAGTAAACCCCG 
                   
                 LFQSDVMPFLKEKQRSLLY 
               
               
                   
                   
                   
                 CGGCGTATAGCAGAGGTGGCGCACC 
                   
                 YGGGGSGGGGSGGGGSGG 
               
               
                   
                   
                   
                 GGTTTATGTGGTGGCTGAATCAGCT 
                   
                 GGSKFGLFFLNFINSTTVQE 
               
               
                   
                   
                   
                 TCGACGACTGAGTGGGCTGCTCAAT 
                   
                 QSIVRMQEITEYVDKLNFE 
               
               
                   
                   
                   
                 TTGGCCTACCGATGATATTAAGTTG 
                   
                 QILVYENHFSDNGVVGAPL 
               
               
                   
                   
                   
                 GATTATAAATACTAACGAAAAGAAA 
                   
                 TVSGFLLGLTEKIKIGSLNHI 
               
               
                   
                   
                   
                 GCACAACTTGAGCTTTATAATGAAG 
                   
                 ITTHHPVRIAEEACLLDQLS 
               
               
                   
                   
                   
                 TGGCTCAAGAATATGGGCACGATAT 
                   
                 EGRFILGFSDCEKKDEMHF 
               
               
                   
                   
                   
                 TCATAATATCGACCATTGCTTATCAT 
                   
                 FNRPVEYQQQLFEECYEIIN 
               
               
                   
                   
                   
                 ATATAACATCTGTAGATCATGACTC 
                   
                 DALTTGYCNPDNDFYSFPK 
               
               
                   
                   
                   
                 AATTAAAGCGAAAGAGATTTGCCGG 
                   
                 ISVNPHAYTPGGPRKYVTA 
               
               
                   
                   
                   
                 AAATTTCTGGGGCATTGGTATGATT 
                   
                 TSHHIVEWAAKKGIPLIFK 
               
               
                   
                   
                   
                 CTTATGTGAATGCTACGACTATTTTT 
                   
                 WDDSNDVRYEYAERYKAV 
               
               
                   
                   
                   
                 GATGATTCAGACCAAACAAGAGGTT 
                   
                 ADKYDVDLSEIDHQLMILV 
               
               
                   
                   
                   
                 ATGATTTCAATAAAGGGCAGTGGCG 
                   
                 NYNEDSNKAKQETRAFISD 
               
               
                   
                   
                   
                 TGACTTTGTATTAAAAGGACATAAA 
                   
                 YVLEMHPNENFENKLEEIIA 
               
               
                   
                   
                   
                 GATACTAATCGCCGTATTGATTACA 
                   
                 ENAVGNYTECITAAKLAIE 
               
               
                   
                   
                   
                 GTTACGAAATCAATCCCGTGGGAAC 
                   
                 KCGAKSVLLSFEPMNDLMS 
               
               
                   
                   
                   
                 GCCGCAGGAATGTATTGACATAATT 
                   
                 QKNVINIVDDNIKKYHTEY 
               
               
                   
                   
                   
                 CAAAAAGACATTGATGCTACAGGAA 
                   
                 T* 
               
               
                   
                   
                   
                 TATCAAATATTTGTTGTGGATTTGAA 
                   
                   
               
               
                   
                   
                   
                 GCTAATGGAACAGTAGACGAAATTA 
                   
                   
               
               
                   
                   
                   
                 TTGCTTCCATGAAGCTCTTCCAGTCT 
                   
                   
               
               
                   
                   
                   
                 GATGTCATGCCATTTCTTAAAGAAA 
                   
                   
               
               
                   
                   
                   
                 AACAACGTTCGCTATTATATTATGG 
                   
                   
               
               
                   
                   
                   
                 CGGTGGCGGTAGCGGCGGTGGCGGT 
                   
                   
               
               
                   
                   
                   
                 AGCGGCGGTGGCGGTAGCGGCGGTG 
                   
                   
               
               
                   
                   
                   
                 GCGGTAGCAAATTTGGATTGTTCTTC 
                   
                   
               
               
                   
                   
                   
                 CTTAACTTCATCAATTCAACAACTGT 
                   
                   
               
               
                   
                   
                   
                 TCAAGAACAGAGTATAGTTCGCATG 
                   
                   
               
               
                   
                   
                   
                 CAGGAAATAACGGAGTATGTTGATA 
                   
                   
               
               
                   
                   
                   
                 AGTTGAATTTTGAACAGATTTTAGT 
                   
                   
               
               
                   
                   
                   
                 GTATGAAAATCATTTTTCAGATAAT 
                   
                   
               
               
                   
                   
                   
                 GGTGTTGTCGGCGCTCCTCTGACTGT 
                   
                   
               
               
                   
                   
                   
                 TTCTGGTTTTCTGCTCGGTTTAACAG 
                   
                   
               
               
                   
                   
                   
                 AGAAAATTAAAATTGGTTCATTAAA 
                   
                   
               
               
                   
                   
                   
                 TCACATCATTACAACTCATCATCCTG 
                   
                   
               
               
                   
                   
                   
                 TCCGCATAGCGGAGGAAGCTTGCTT 
                   
                   
               
               
                   
                   
                   
                 ATTGGATCAGTTAAGTGAAGGGAGA 
                   
                   
               
               
                   
                   
                   
                 TttattTTAGGGTTTAGTGATTGCGA 
                   
                   
               
               
                   
                   
                   
                 AAAAAAAGATGAAATGCATTTTTTT 
                   
                   
               
               
                   
                   
                   
                 AATCGCCCGGTTGAATATCAACAGC 
                   
                   
               
               
                   
                   
                   
                 AACTATTTGAAGAGTGTTATGAAAT 
                   
                   
               
               
                   
                   
                   
                 CATTAACGATGCTTTAACAACAGGC 
                   
                   
               
               
                   
                   
                   
                 TATTGTAATCCAGATAACGATTTTTA 
                   
                   
               
               
                   
                   
                   
                 TAGCTTCCCTAAAATATCTGTAAATC 
                   
                   
               
               
                   
                   
                   
                 CCCATGCTTATACGCCAGGCGGACC 
                   
                   
               
               
                   
                   
                   
                 TCGGAAATATGTAACAGCAACCAGT 
                   
                   
               
               
                   
                   
                   
                 CATCATATTGTTGAGTGGGCGGCCA 
                   
                   
               
               
                   
                   
                   
                 AAAAAGGTATTCCTCTCATCTTTAA 
                   
                   
               
               
                   
                   
                   
                 GTGGGATGATTCTAATGATGTTAGA 
                   
                   
               
               
                   
                   
                   
                 TATGAATATGCTGAAAGATATAAAG 
                   
                   
               
               
                   
                   
                   
                 CCGTTGCGGATAAATATGACGTTGA 
                   
                   
               
               
                   
                   
                   
                 CCTATCAGAGATAGACCATCAGTTA 
                   
                   
               
               
                   
                   
                   
                 ATGATATTAGTTAACTATAACGAAG 
                   
                   
               
               
                   
                   
                   
                 ATAGTAATAAAGCTAAACAAGAGAC 
                   
                   
               
               
                   
                   
                   
                 GCGTGCATTTATTAGTGATTATGTTC 
                   
                   
               
               
                   
                   
                   
                 TTGAAATGCACCCTAATGAAAATTT 
                   
                   
               
               
                   
                   
                   
                 CGAAAATAAACTTGAAGAAATAATT 
                   
                   
               
               
                   
                   
                   
                 GCAGAAAACGCTGTCGGAAATTATA 
                   
                   
               
               
                   
                   
                   
                 CGGAGTGTATAACTGCGGCTAAGTT 
                   
                   
               
               
                   
                   
                   
                 GGCAATTGAAAAGTGTGGTGCGAAA 
                   
                   
               
               
                   
                   
                   
                 AGTGTATTGCTGTCCTTTGAACCAAT 
                   
                   
               
               
                   
                   
                   
                 GAATGATTTGATGAGCCAAAAAAAT 
                   
                   
               
               
                   
                   
                   
                 GTAATCAATATTGTTGATGATAATA 
                   
                   
               
               
                   
                   
                   
                 TTAAGAAGTACCACACGGAATATAC 
                   
                   
               
               
                   
                   
                   
                 CTAA 
                   
                   
               
               
                   
               
               
                 GOI 
                 SpecR 
                 20 
                 ATGAGGGAAGCGGTGATCGCCGAA 
                 31 
                 MREAVIAEVSTQLSEVVGV 
               
               
                   
                   
                   
                 GTATCGACTCAACTATCAGAGGTAG 
                   
                 IERHLEPTLLAVHLYGSAV 
               
               
                   
                   
                   
                 TTGGCGTCATCGAGCGCCATCTCGA 
                   
                 DGGLKPH SDIDLLVTVTVR 
               
               
                   
                   
                   
                 ACCGACGTTGCTGGCCGTACATTTG 
                   
                 LDETTRRALINDLLETSASP 
               
               
                   
                   
                   
                 TACGGCTCCGCAGTGGATGGCGGCC 
                   
                 GESEILRAVEVTIVVHDDIIP 
               
               
                   
                   
                   
                 TGAAGCCACACAGTGATATTGATTT 
                   
                 WRYPAKRELQFGEWQRND 
               
               
                   
                   
                   
                 GCTGGTTACGGTGACCGTAAGGCTT 
                   
                 ILAGIFEPATIDIDLAILLTK 
               
               
                   
                   
                   
                 GATGAAACAACGCGGCGAGCTTTGA 
                   
                 AREHSVALVGPAAEELFDP 
               
               
                   
                   
                   
                 TCAACGACCTTTTGGAAACTTCGGC 
                   
                 VPEQDLFEALNETLTLWNS 
               
               
                   
                   
                   
                 TTCCCCTGGAGAGAGCGAGATTCTC 
                   
                 PPDWAGDERNVVLTLSRIW 
               
               
                   
                   
                   
                 CGCGCTGTAGAAGTCACCATTGTTG 
                   
                 YSAVTGKIAPKDVAADWA 
               
               
                   
                   
                   
                 TGCACGACGACATCATTCCGTGGCG 
                   
                 MERLPAQYQPVILEARQAY 
               
               
                   
                   
                   
                 TTATCCAGCTAAGCGCGAACTGCAA 
                   
                 LGQEEDRLASRADQLEEFV 
               
               
                   
                   
                   
                 TTTGGAGAATGGCAGCGCAATGACA 
                   
                 HYVKGEITKVVGK* 
               
               
                   
                   
                   
                 TTCTTGCAGGTATCTTCGAGCCAGCC 
                   
                   
               
               
                   
                   
                   
                 ACGATCGACATTGATCTGGCTATCTT 
                   
                   
               
               
                   
                   
                   
                 GCTGACAAAAGCAAGAGAACATAG 
                   
                   
               
               
                   
                   
                   
                 CGTTGCCTTGGTAGGTCCAGCGGCG 
                   
                   
               
               
                   
                   
                   
                 GAGGAACTCTTTGATCCGGTTCCTG 
                   
                   
               
               
                   
                   
                   
                 AACAGGATCTATTTGAGGCGCTAAA 
                   
                   
               
               
                   
                   
                   
                 TGAAACCTTAACGCTATGGAACTCG 
                   
                   
               
               
                   
                   
                   
                 CCGCCCGACTGGGCTGGCGATGAGC 
                   
                   
               
               
                   
                   
                   
                 GAAATGTAGTGCTTACGTTGTCCCG 
                   
                   
               
               
                   
                   
                   
                 CATTTGGTACAGCGCAGTAACCGGC 
                   
                   
               
               
                   
                   
                   
                 AAAATCGCGCCGAAGGATGTCGCTG 
                   
                   
               
               
                   
                   
                   
                 CCGACTGGGCAATGGAGCGCCTGCC 
                   
                   
               
               
                   
                   
                   
                 GGCCCAGTATCAGCCCGTCATACTT 
                   
                   
               
               
                   
                   
                   
                 GAAGCTAGACAGGCTTATCTTGGAC 
                   
                   
               
               
                   
                   
                   
                 AAGAAGAAGATCGCTTGGCCTCGCG 
                   
                   
               
               
                   
                   
                   
                 CGCAGATCAGTTGGAAGAATTTGTC 
                   
                   
               
               
                   
                   
                   
                 CACTACGTGAAAGGCGAGATCACCA 
                   
                   
               
               
                   
                   
                   
                 AGGTAGTCGGCAAATGA 
               
               
                   
               
               
                   ‡ RBS designed computationally using the Ribosome Binding Site Calculator. 58   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Primers used to assemble the bacterial two-hybrid system. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 F Primer 
                   
                 R Primer 
                   
               
               
                   
                 SEQ 
                   
                 SEQ 
                   
               
               
                 Component 
                 ID NO: 
                 F Primer 
                 ID NO: 
                 R Primer 
               
               
                   
               
               
                 RpoZ/HA4 with 
                 32 
                 GTGCAGTAAGGAGGAAAAAA 
                 54 
                 GTCAGGGGCGGGGTTTTTTTT 
               
               
                 pAB078d8 
                   
                   
                   
                 TAGGGCCCTACTGACTGTTAG 
               
               
                 overhangs 
                   
                   
                   
                 CAGGTGCGGTAATTGA 
               
               
                   
               
               
                 pAB078d8 with 
                 33 
                 CAGTCAGTAGGGCCCTAAAA 
                 55 
                 CACAGTTCTCGTCATCAGCTC 
               
               
                 RpoZ/HA4 
                   
                   
                   
                 TCTGGTTGCTTTAGCTAATAC 
               
               
                 overhang piece 1 
                   
                   
                   
                 ACCATAAGCATTTTCC 
               
               
                   
               
               
                 pAB078d8 with 
                 34 
                 TAGCTAAAGCAACCAGAGAG 
                 56 
                 CAGTTACGCGTGCCATTTTTT 
               
               
                 RpoZ/HA4 
                   
                   
                   
                 TTTCCTCCTTACTGCACTTAG 
               
               
                 overhang piece 2 
                   
                   
                   
                 CGTTTCGGCGCCGGAT 
               
               
                   
               
               
                 Src/CDC37 into 
                 35 
                 CAATTCCCCTCTAGAAATAA 
                 57 
                 GTCAGGGGCGGGGTTTTTTTT 
               
               
                 pAB078d8 
                   
                 TTTTG 
                   
                 TAGGGCCCTACTGACTGTTAC 
               
               
                   
                   
                   
                   
                 ACACTGACATCCTTCTCATCG 
               
               
                   
               
               
                 Insulin Receptor 
                 36 
                 CGCTGTAGAGAAAATTGGTA 
                 58 
                 CAGGGGCGGGGTTTTTTTTTA 
               
               
                 Substrate RpoZ 
                   
                   
                   
                 GGGCCCTACTGACTGTTATTA 
               
               
                 fusion into 
                   
                   
                   
                 GCCAAGATCCATCTTCA 
               
               
                 pAB078d8* 
                   
                   
                   
                   
               
               
                   
               
               
                 Insulin Receptor 
                 37 
                 GACGCGGAATGGTACTGGGG 
                 59 
                 GTTACGCGTGCCATTTTTTTT 
               
               
                 SH2_cI fusion 
                   
                   
                   
                 TCCTCCTTACTGCACTTATTA 
               
               
                 into pAB078d8* 
                   
                   
                   
                 CGAAACCGGATACAACA 
               
               
                   
               
               
                 Src/CDC37 into 
                 38 
                 ATATGGTCTCACATGTCCAA 
                 60 
                 ATATGGTCTCATTTACACACT 
               
               
                 pBAD33t 
                   
                 GCCGCAGACTCAG 
                   
                 GACATCCTTCTCATCG 
               
               
                   
               
               
                 RpoZ/pl30cas 
                 39 
                 ATATGGTCTCACATGGCACG 
                 61 
                 ATATGGTCTCATTTACCCCTG 
               
               
                 substrate into 
                   
                 CGTAACTGTTC 
                   
                 TAGGTGGACG 
               
               
                 pBAD33t 
                   
                   
                   
                   
               
               
                   
               
               
                 cI/SH2 into 
                 40 
                 ATATGGTCTCACATGAGTAT 
                 62 
                 ATATGGTCTCATTTAGCAGAC 
               
               
                 pBAD33t 
                   
                 CAGCAGCAGGGTAAAAAG 
                   
                 GTTGGTCAGGC 
               
               
                   
               
               
                 pB2H 1b  Gibson 
                 41 
                 ATGACTACGTCCACCTACAG 
                 63 
                 AAGATAAAAAGAATAGATCCC 
               
               
                 piece 1 
                   
                 GGGTAATAACAATTCCCCTC 
                   
                 AGCCCTGTGTATAACTCACTA 
               
               
                   
                   
                 TAGAAATAATTTTGTTTAAC 
                   
                 CTTTAGTCAGTTCCGCA 
               
               
                   
               
               
                 pB2H 1b  Gibson 
                 42 
                 TGAGTTATACACAGGGCTGG 
                 64 
                 CCCCTGTAGGTGGACGTAGTC 
               
               
                 piece 2 
                   
                   
                   
                 ATAGTCCTCCATCCACGCAGC 
               
               
                   
                   
                   
                   
                 TGCACGACGA 
               
               
                   
               
               
                 pB2H 1b  Gibson 
                 43 
                 GTGCAGTAAGGAGGAAAAAA 
                 65 
                 GCCCATGGTATATCTCCTTCT 
               
               
                 piece 3 
                   
                 AATGGC 
                   
                 TAAAGT 
               
               
                   
               
               
                 pB2H 1b  Gibson 
                 44 
                 TAAAATTCGTAGACTACAAG 
                 66 
                 ACAGTTACGCGTGCCATTTTT 
               
               
                 piece 4 
                   
                 GACGACGATGACAAGTGGTA 
                   
                 TTTTCCTCCTTACTGCACTTA 
               
               
                   
                   
                 TTTTGGGAAGATCACTCGT 
                   
                 GCAGACGTTGGTCAGGC 
               
               
                   
               
               
                 B2H ShcA 
                 45 
                 TAATAACAATTCCCCTCTAG 
                 67 
                 GGGAATTGTTATTAGCCCGGA 
               
               
                 substrate 
                   
                 AAATAATTTTGTTTAACTTT 
                   
                 AAATCGTTATAATACTGATGA 
               
               
                   
                   
                 AAG 
                   
                 TCCGCAGCTGCACGACG 
               
               
                   
               
               
                 B2H EGFR 
                 45 
                 TAATAACAATTCCCCTCTAG 
                 68 
                 GGGAATTGTTATTAATCGCCC 
               
               
                 Substrate 
                   
                 AAATAATTTTGTTTAACTTT 
                   
                 TGAATCACCAGATAGCGCTGC 
               
               
                   
                   
                 AAG 
                   
                 GGCGCAGCTGCACGACG 
               
               
                   
               
               
                 B2H MidT 
                 45 
                 TAATAACAATTCCCCTCTAG 
                 69 
                 GAATTGTTATTACAGATAAAT 
               
               
                 Substrate 
                   
                 AAATAATTTTGTTTAACTTT 
                   
                 CGGAATTTCTTCATACTGCGG 
               
               
                   
                   
                 AAG 
                   
                 TTCCGCAGCTGCACGACG 
               
               
                   
               
               
                 BB034 PTP1B 1-321   
                 46 
                 GTCAGTGTGTAAGTGCAGAA 
                 70 
                 CTCATCCGCCAAAACAGCCTC 
               
               
                 into pBAD 1c   
                   
                 AGAGGAGAAATACTAGATGG 
                   
                 AATTGTGTGGCTCCAGGATTC 
               
               
                   
                   
                 AGATGGAAAAGGAGTTCGAG 
                   
                 G 
               
               
                   
               
               
                 BB034 
                 47 
                 TAATCTAGAGAAAGAGGAGA 
                 71 
                 TTACACACTGACATCCTTCTC 
               
               
                 Src/CDC37 
                   
                 AATACTAGATGTCCAAGCCG 
                   
                 ATCG 
               
               
                   
                   
                 CAGACTC 
                   
                   
               
               
                   
               
               
                 ProD into B2H 
                 48 
                 CTCTAGTAAAAGTTAAACAA 
                 72 
                 TTCTAGAGCACAGCTAACACC 
               
               
                   
                   
                 AATTATTTGTAGAGGG 
                   
                 AC 
               
               
                   
               
               
                 ProD Overhang 
                 49 
                 AACTTTTACTAGAGGAATTC 
                 63 
                 AAGATAAAAAGAATAGATCCC 
               
               
                 ProRBS 
                   
                 GAGCTCTTAAAGAGGAGAAA 
                   
                 AGCCCTGTGTATAACTCACTA 
               
               
                 Src/CDC37 
                   
                 GGTCATGGGCTCCAAGCCGC 
                   
                 CTTTAGTCAGTTCCGCA 
               
               
                   
               
               
                 Sal28 RBS 
                 50 
                 AACTTTTACTAGAGCGAAAA 
                 73 
                 GAACCAATGAATGATTTGATG 
               
               
                 Src/CDC37 
                   
                 AAAGTAAGGCGGTAATCCAT 
                   
                 AGC 
               
               
                   
                   
                 GGGCTCCAAGCCGC 
                   
                   
               
               
                   
               
               
                 BB030 PTP1B 
                 51 
                 AGTGTGTAAGTGCAGATTAA 
                 74 
                 GTTTTTTTTTAGGGCCCTACT 
               
               
                 into pB2H S1.2Sal28   
                   
                 AGAGGAGAAATACTAGATGG 
                   
                 GACTGTCAATTGTGTGGCTCC 
               
               
                   
                   
                 AGATGGAAAAGGAGTTCGAG 
                   
                 AGGATTC 
               
               
                   
               
               
                 BB034 PTP1B 
                 52 
                 TCAGTGTGTAAGTGCAGTCA 
                 74 
                 GTTTTTTTTTAGGGCCCTACT 
               
               
                 into pB2H 1.2Sal28   
                   
                 CACAGGAAAGTACTAGATGG 
                   
                 GACTGTCAATTGTGTGGCTCC 
               
               
                   
                   
                 AGATGGAAAAGGAGTTCGAG 
                   
                 AGGATTC 
               
               
                   
               
               
                 B2H Swap 
                 53 
                 GCGTACATTGGCTCCGTTCA 
                 75 
                 GACCTGCAGATTAAAGAGGGA 
               
               
                 LuxAB/SpecR 
                   
                 TTTGCCGACTACCTTGGTGA 
                   
                 AAAATGAGGGAAGCGGTGATC 
               
               
                   
                   
                 TC 
                   
                 G 
               
               
                   
               
               
                 *Insulin receptor substrate/SH2 domains 59  were used initially, but failed to activate the 
               
               
                 operon (data not shown) 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Primers used to assemble pathways for terpenoid biosynthesis. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 F Primer 
                   
                 R Primer 
                   
               
               
                 Component 
                 SEQ ID NO: 
                 F Primer 
                 SEQ ID NO: 
                 R Primer 
               
               
                   
               
               
                 GGPPS into 
                 76 
                 TATTGAGCTCCACCGCGGA 
                 80 
                 TATTGTCGACTTATTTATTAC 
               
               
                 pTrc99t 
                   
                 GGAGGAATG 
                   
                 GCTGGATGATGTAGTC 
               
               
                   
               
               
                 TXS into pTrc99t 
                 77 
                 TATTGGTCTCCCATGAGCA 
                 81 
                 TATTGGTCTCCGTCCTTCCAA 
               
               
                   
                   
                 GCAGCACTGGCAC 
                   
                 CGCATTCAACATGTTG 
               
               
                   
               
               
                 ABS into pTrc99t 
                 78 
                 ATAAAGGTCTCCCATGGTG 
                 82 
                 TATTAGGTCTCGAGCTCTTAG 
               
               
                   
                   
                 AAACGAGAATTTCCTCCAG 
                   
                 GCAACTGGTTGGAAGAGGC 
               
               
                   
               
               
                 pMBIS TetR- 
                 79 
                 AGATCACTACCGGGCGTAT 
                 83 
                 GCCGCCGGCTTCCATTTATTA 
               
               
                 &gt;CmR 
                   
                 TTTTTGAGTTATCGAGATT 
                   
                 CGCCCCGCCCTG 
               
               
                   
                   
                 TTCAGGAGCTAAGGAAGCT 
                   
                   
               
               
                   
                   
                 AAAATGGAGAAAAAAATCA 
                   
                   
               
               
                   
                   
                 CTGGATATACCAC 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Primers used for site-directed mutagenesis. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 F Primer 
                   
                 R Primer 
                   
               
               
                 Mutant 
                 SEQ ID NO: 
                 F Primer 
                 SEQ ID NO: 
                 R Primer 
               
               
                   
               
               
                 PTP1B 
                  84 
                 GTCCAGTACTTTATTGGGGTT 
                 107 
                 ATCTCGGACATGCTCAGTTCC 
               
               
                 (C215S) 
                   
                 CAGGCGGATGGAACTGAGCAT 
                   
                 ATCCGCCTGAACCCCAATAAA 
               
               
                   
                   
                 GTCCGAGAT 
                   
                 GTACTGGAC 
               
               
                   
               
               
                 ABS 
                  85 
                 GAGAGAGAATCCTGTTCCTAG 
                 108 
                 GAAGGCCCATGGCTGTATCCG 
               
               
                 (D404A) 
                   
                 TATTGCGGATACAGCCATGGG 
                   
                 CAATATCAGGAACAGGATTCT 
               
               
                   
                   
                 CCTTC 
                   
                 CTCTC 
               
               
                   
               
               
                 ABS 
                  86 
                 ACAAAAACTTCCAATTTCACT 
                 109 
                 CCATGGGCGTCATAAAGATCC 
               
               
                 (D621A) 
                   
                 GTTATTTTAGCGGATCTTTAT 
                   
                 GCTAAAATAACAGTGAAATTG 
               
               
                   
                   
                 GACGCCCATGG 
                   
                 GAAGTTTTTGT 
               
               
                   
               
               
                 ADS 
                  87 
                 CGTAAGCATCGTAAGTGTCCG 
                 110 
                 GCTGTTATCACCCTGATCGCG 
               
               
                 (D299A) 
                   
                 CGATCAGGGTGATAACAGC 
                   
                 GACACTTACGATGCTTACG 
               
               
                   
               
               
                 GHS 
                  88 
                 CCCATGCGTGTCGTATAAGTC 
                 111 
                 CGATCTTGATGACAATGTTAG 
               
               
                 (D343A) 
                   
                 CGCTAACATTGTCATCAAGAT 
                   
                 CGGACTTATACGACACGCATG 
               
               
                   
                   
                 CG 
                   
                 GG 
               
               
                   
               
               
                 GHS 
                  89 
                 CAATGGCACCCCCAACNNKGG 
                 112 
                 GTTGGGGGTGCCATTGTTC 
               
               
                 (T455X) 
                   
                 TATGTGTGTACTTAATCTGAT 
                   
                   
               
               
                   
                   
                 CCCG 
                   
                   
               
               
                   
               
               
                 GHS 
                  90 
                 CAACACCGGTATGTGTGTANN 
                 113 
                 TACACACATACCGGTGTTGGG 
               
               
                 (L450X) 
                   
                 KAATCTGATCCCGTTGCTGCT 
                   
                   
               
               
                   
                   
                 TATG 
                   
                   
               
               
                   
               
               
                 GHS 
                  91 
                 AAACGCTTGGGAACGCNNKCT 
                 114 
                 GCGTTCCCAAGCGTTTTTG 
               
               
                 (Y415X) 
                   
                 GGAAGCGTATTTGCAGGATG 
                   
                   
               
               
                   
               
               
                 GHS 
                  92 
                 CTTCTGGATGGCCGCGNNKAT 
                 115 
                 CGCGGCCATCCAGAAGT 
               
               
                 (A319X) 
                   
                 TTCAGAACCAGAATTTAGTGG 
                   
                   
               
               
                   
                   
                 CTC 
                   
                   
               
               
                   
               
               
                 GHS 
                  93 
                 ACCATCTGATTGAACTGGCTN 
                 116 
                 AGCCAGTTCAATCAGATGGTG 
               
               
                 (S484X) 
                   
                 NKCGACTGGTCGATGATGCGA 
                   
                 G 
               
               
                   
                   
                 G 
                   
                   
               
               
                   
               
               
                 GHS 
                  94 
                 CGTCCTGGCGCGGNNKATTCA 
                 117 
                 CCGCGCCAGGACGTG 
               
               
                 (S561X) 
                   
                 GTTTATGTATAACCAGGGGGA 
                   
                   
               
               
                   
                   
                 C 
                   
                   
               
               
                   
               
               
                 ADS 
                  95 
                 CAACTGCGGTAAAGAGTTTGT 
                 118 
                 TTCTTTAACAAACTCTTTACC 
               
               
                 (F370X) 
                   
                 TAAAGAANNKGTACGTAACCT 
                   
                 GCAGTTG 
               
               
                   
                   
                 GATGGTTGAAGC 
                   
                   
               
               
                   
               
               
                 ADS 
                  96 
                 CATGACCCGGTTGTTATCATC 
                 119 
                 GGTGATGATAACAACCGGGTC 
               
               
                 (G400X) 
                   
                 ACCNNKGGTGCAAACCTGCTG 
                   
                 ATG 
               
               
                   
                   
                 ACCAC 
                   
                   
               
               
                   
               
               
                 ADS 
                  97 
                 CCGGCGGTGCAAACCTGNNKA 
                 120 
                 CAGGTTTGCACCGCCGG 
               
               
                 (L405X) 
                   
                 CCACCACTTGCTATCTGGG 
                   
                   
               
               
                   
               
               
                 ADS 
                  98 
                 CTGTTCCGTTACTCCGGTATT 
                 121 
                 CAGAATACCGGAGTAACGGAA 
               
               
                 (G439X) 
                   
                 CTGNNKCGTCGTCTGAACGAC 
                   
                 CAG 
               
               
                   
                   
                 CTGATG 
                   
                   
               
               
                   
               
               
                 ADS 
                  99 
                 GGCAGTAATCTACCTGTGCCA 
                 122 
                 CTGGCACAGGTAGATTACTGC 
               
               
                 (F514X) 
                   
                 GNNKCTGGAAGTACAGTACGC 
                   
                 C 
               
               
                   
                   
                 TGGTAAAG 
                   
                   
               
               
                   
               
               
                 MidT 
                 100 
                 CAGCTGCGGAACCGCAGTTTG 
                 123 
                 ATCGGAATTTCTTCAAACTGC 
               
               
                 Substrate 
                   
                 AAGAAATTCCGAT 
                   
                 GGTTCCGCAGCTG 
               
               
                 (Y/F) 
                   
                   
                   
                   
               
               
                   
               
               
                 p130Cas 
                 101 
                 TGGATGGAGGACTTTGACTTC 
                 124 
                 GTCAAAGTCCTCCATCCACGC 
               
               
                 Substrate 
                   
                 GTCCACCTACAGGGGTAATAA 
                   
                 AGCTGCACGACG 
               
               
                 (Y/F) 
                   
                 CAATTC 
                   
                   
               
               
                   
               
               
                 SH2 
                 102 
                 CTCTCCGTTTCTGACTTTGAC 
                 125 
                 AAGTCAGAAACGGAGAGGGCA 
               
               
                 (Superbinder 
                   
                 AACGCCAAGGGGCTCAATGTG 
                   
                 TAGGCACCTTTTACCGTCTCG 
               
               
                 mutations) 
                   
                 CTGCACTACAAGATCCGCAAG 
                   
                 CTCTCCCG 
               
               
                   
                   
                 CTG 
                   
                   
               
               
                   
               
               
                 SH2 
                 103 
                 AAACACTACCTGATCCGCAAG 
                 126 
                 GCTGTCCAGCTTGCGGATCAG 
               
               
                 (L13K 
                   
                 CTGGACAGC 
                   
                 GTAGTGTTTCACATTGAGCCC 
               
               
                 K15L)* 
                   
                   
                   
                 CTTGGC* 
               
               
                   
               
               
                 pTrc99a 
                 104 
                 TATTGGTCTCTCGCGGTATCA 
                 127 
                 TATTGGTCTCAGTGACCCCAC 
               
               
                 (remove 
                   
                 TTGCAGCAC 
                   
                 ACTACCATCGG 
               
               
                 BsaI sites) 
                   
                   
                   
                   
               
               
                 piece 1 
                   
                   
                   
                   
               
               
                   
               
               
                 pTrc99a 
                 105 
                 TATTGGTCTCATCACCCCATG 
                 128 
                 TATTGGTCTCACGCGTGACCC 
               
               
                 (remove 
                   
                 CGAGAGTAGG 
                   
                 ACGCTCACCG 
               
               
                 BsaI sites) 
                   
                   
                   
                   
               
               
                 piece 2 
                   
                   
                   
                   
               
               
                   
               
               
                 ADS ep 
                 106 
                 AACAATTTCACACAGGAAACA 
                 129 
                 GCCTGCAGGTCGACTCTAGA 
               
               
                 PCR 
                   
                 GACC 
                   
                   
               
               
                   
               
               
                 *The original superbinder primer mutated the incorrect lysine residue (13 vs. 15). This 
               
               
                 primer corrects that error. The residue numbering system used for this protein matches 
               
               
                 that of Kaneko et. al. 40   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Gene sources. 
               
            
           
           
               
               
               
               
            
               
                 Component 
                 Organism 
                 Plasmid 
                 Source 
               
               
                   
               
               
                 Src 
                 
                   H. sapiens 
                 
                 pDONR223_ 
                 Addgene: 82165 
               
               
                   
                   
                 SRC_WT 
                   
               
               
                 CDC37 
                 
                   H. sapiens 
                 
                 pBACgus4x/ 
                 Addgene: 40398 
               
               
                   
                   
                 cdc37/RocCOR 
                   
               
               
                   
                   
                 LRRK2  
                   
               
               
                   
                   
                 1867-2176 
                   
               
               
                 PTP1B 
                 
                   H. sapiens 
                 
                 pET21B_ 
                 Nicholas  
               
               
                   
                   
                 PTP1B 
                 Tonks, Cold 
               
               
                   
                   
                   
                 Spring Harbor 
               
               
                 TC-PTP 
                 
                   H. sapiens 
                 
                 pBG100- 
                 Addgene: 33365 
               
               
                   
                   
                 TCPTP 
                   
               
               
                 PTPN6 
                 
                   H. sapiens: 
                 
                 pGEX-2T  
                 Addgene: 8594 
               
               
                   
                   
                 SHP1 WT 
                   
               
               
                 PTPN12 
                 
                   H. sapiens 
                 
                 DONR223_ 
                 Addgene: 81528 
               
               
                   
                   
                 PTPN12_ 
                   
               
               
                   
                   
                 p.E57D 
                   
               
               
                 LuxAB 
                   
                 pAB078d8 
                 Addgene: 79206 
               
               
                 RpoZ 
                 
                   Escherichia coli 
                 
                 pAB094a 
                 Addgene: 79241 
               
               
                 cI434 
                 
                   Escherichia  
                 
                 pAB078d8 
                 Addgene: 79206 
               
               
                   
                 
                   virus Lambda 
                 
                   
                   
               
               
                 SH2 
                 
                   Rous sarcoma  
                 
                 Kras-SRC  
                 Addgene: 78302 
               
               
                   
                 
                   virus 
                 
                 FRET  
                   
               
               
                   
                   
                 Biosensor 
                   
               
               
                 p130cas 
                 
                   H. sapiens 
                 
                 Synthetic 
                 Integrated DNA 
               
               
                   
                   
                   
                 Technologies, Inc. 
               
               
                 midT 
                 
                   H. sapiens 
                 
                 Synthetic 
                 Integrated DNA 
               
               
                   
                   
                   
                 Technologies, Inc. 
               
               
                 EGFR 
                 
                   H. sapiens 
                 
                 Synthetic 
                 Integrated DNA 
               
               
                   
                   
                   
                 Technologies, Inc. 
               
               
                 ShcA 
                 
                   H. sapiens 
                 
                 Synthetic 
                 Integrated DNA 
               
               
                   
                   
                   
                 Technologies, Inc. 
               
               
                 MBIS 
                 
                   S. cerevisiae 
                 
                 pMBIS 
                 Addgene: 17817 
               
               
                 ADS 
                 
                   Artemisia  
                 
                 pADS 
                 Addgene: 19040 
               
               
                   
                 
                   annua 
                 
                   
                   
               
               
                 GHS 
                 
                   Abies grandis 
                 
                 pTrcHUM 
                 Addgene: 19003 
               
               
                 ABS 
                 
                   Abies grandis 
                 
                 pSBET/AgAs 
                 Reuben Peters,  
               
               
                   
                   
                   
                 Iowa State 
               
               
                   
                   
                   
                 University 
               
               
                 TXS 
                 
                   Taxus brevifola 
                 
                 M60 
                 David W.  
               
               
                   
                   
                   
                 Christianson, 
               
               
                   
                   
                   
                 University of 
               
               
                   
                   
                   
                 Pennsylvania 
               
               
                 ABA 
                 
                   Abies grandis 
                 
                 pTrc99a 
                 Addgene: 35153 
               
               
                 GGPPS 
                 
                   Taxus  
                 
                 gBlock 
                 Integrated DNA 
               
               
                   
                 
                   canadensis 
                 
                   
                 Technologies, Inc. 
               
               
                 A0A166A5J3 
                 
                   S. Suecicum  
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                   
                 HHB10207 ss-3 
                   
                   
               
               
                 A0A0D9X487 
                 
                   L. perrieri 
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                 F2DRF1 
                 
                   H. vulgare 
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                 A2XI80 
                 
                   O. sativa 
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                 A0A0D9ZGD1 
                 
                   O. glumipatula 
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                 A0A0K9RZT8 
                 
                   S. olaracea 
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                 A0A1I1AC30 
                 
                   A.aquimarinus 
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                 A0A1S3XW43 
                 
                   N. tabacum 
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                 A0A0D3D8G7 
                 
                   B. oleracea 
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                 B9IF04 
                 
                   P. trichocarpa 
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                 A0A067L3D3 
                 
                   J. curcas 
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                 A0A0C2TFL3 
                 
                   A.Muscaria  
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                   
                   Koide  BX008 
                   
                   
               
               
                 A0A022S1C8 
                 
                   E. guttata 
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                 G4TNA6 
                 
                   S. indica 
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                 A0A1L7WMZ8 
                 
                   P. subalpine 
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                 A0A078IZJ5 
                 
                   B. napus 
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                 A0A0C9VSL7 
                 
                   S. stellatus  
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                   
                 SS14 
                   
                   
               
               
                 G2QRS0 
                 
                   T. terrestris  
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                   
                 ATCC 38088 
                   
                   
               
               
                 A0A2H3DKU3 
                 
                   A. 
                   gallica 
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                 A0A0D2L718 
                 
                   H. sublateritium  
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                   
                 FD-334 SS-4 
                   
                   
               
               
                 S9Q0922 
                 
                   C. Fuscus  
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                   
                 DSM 2262 
                   
                   
               
               
                 T1LTV1 
                 
                   T. urartu 
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                 A0A287XU99 
                 
                   H. vulgare 
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                 A0A0G2ZSL3 
                 
                   A. 
                   gephyra 
                 
                 Synthetic 
                 Twist Bioscience 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Plasmids 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Anti- 
                 Avail- 
               
               
                 Plasmid 
                 Description 
                 biotic* 
                 ability 
               
               
                   
               
               
                 F-plasmid 
                 The F-plasmid from the  
                 T 
                 AG:  
               
               
                   
                 S1030 strain of  E. coli.   
                   
                 105063 *   *   
               
               
                 pB2H 1b   
                 An early version of B2H that  
                 K 
                 Fox Lab 
               
               
                   
                 lacks PTP1B and contains 
                   
                   
               
               
                   
                 LuxAB as the GOI. 
                   
                   
               
               
                 pBAD 1b.Src   
                 Enables inducible expression  
                 P 
                 Fox Lab 
               
               
                   
                 of Src and CDC37 
                   
                   
               
               
                 pBAD 1b.SH2   
                 Enables inducible expression  
                 P 
                 Fox Lab 
               
               
                   
                 of the SH2 domain. 
                   
                   
               
               
                 pBAD 1b.S   
                 Enables inducible expression  
                 P 
                 Fox Lab 
               
               
                   
                 of the substrate domain. 
                   
                   
               
               
                 pBAD 1b.All   
                 Enables inducible expression  
                 P 
                 Fox Lab 
               
               
                   
                 of Src, CDC37, the SH2 
                   
                   
               
               
                   
                 domain, and the substrate  
                   
                   
               
               
                   
                 domain. 
                   
                   
               
               
                 pB2H 1c.p130cas   
                 An early version of B2H that (i)  
                 K 
                 Fox Lab 
               
               
                   
                 lacks PTP1B and Src, (ii) 
                   
                   
               
               
                   
                 contains LuxAB, and (iii)  
                   
                   
               
               
                   
                 includes a substrate from 
                   
                   
               
               
                   
                 p130cas. 
                   
                   
               
               
                 pB2H 1c.midT   
                 An early version of B2H that (i)  
                 K 
                 Fox Lab 
               
               
                   
                 lacks PTP1B and Src, (ii) 
                   
                   
               
               
                   
                 contains LuxAB, and (iii)  
                   
                   
               
               
                   
                 includes a substrate from midT. 
                   
                   
               
               
                 pB2H 1c.ShcA   
                 An early version of B2H that (i)  
                 K 
                 Fox Lab 
               
               
                   
                 lacks PTP1B and Src, (ii) 
                   
                   
               
               
                   
                 contains LuxAB, and (iii)  
                   
                   
               
               
                   
                 includes a substrate from ShCA. 
                   
                   
               
               
                 pB2H 1c.EGFR   
                 An early version of B2H that (i)  
                 K 
                 Fox Lab 
               
               
                   
                 lacks PTP1B and Src, (ii) 
                   
                   
               
               
                   
                 contains LuxAB, and (iii)  
                   
                   
               
               
                   
                 includes a substrate from EGFR. 
                   
                   
               
               
                 pBAD 1d   
                 Enables inducible expression of  
                 P 
                 Fox Lab 
               
               
                   
                 Src and PTP1B. 
                   
                   
               
               
                 pBAD 1d.mut   
                 Enables inducible expression of  
                 P 
                 Fox Lab 
               
               
                   
                 Src and catalytically 
                   
                   
               
               
                   
                 inactive PTP1B (C215S). 
                   
                   
               
               
                 pB2H S1.1Pro1   
                 An early version of B2H that (i)  
                 K 
                 Fox Lab 
               
               
                   
                 lacks PTP1B, (ii) contains 
                   
                   
               
               
                   
                 LuxAB, (iii) places expression  
                   
                   
               
               
                   
                 of Src, CDC37, the SH2 
                   
                   
               
               
                   
                 domain, and the substrate  
                   
                   
               
               
                   
                 domain under control of the 
                   
                   
               
               
                   
                 same Pro1 promoter, and (iv)  
                   
                   
               
               
                   
                 uses the BB034 RBS for Src. 
                   
                   
               
               
                 pB2H S1.1Pro1.   
                 Identical to pB2H S1.1Pro1  except  
                 K 
                 Fox Lab 
               
               
                 
                   mut 
                 
                 for a mutation in the 
                   
                   
               
               
                   
                 substrate (Y4F) 
                   
                   
               
               
                 pB2H S1.1ProD   
                 An early version of B2H that (i)  
                 K 
                 Fox Lab 
               
               
                   
                 lacks PTP1B, (ii) contains 
                   
                   
               
               
                   
                 LuxAB, and (iii) includes the  
                   
                   
               
               
                   
                 ProD promoter and pro RBS 
                   
                   
               
               
                   
                 for Src. 
                   
                   
               
               
                 pB2H S1.1ProD.   
                 Identical to pB2H S1.1ProD  except  
                 K 
                 Fox Lab 
               
               
                 
                   mut 
                 
                 for a mutation in the 
                   
                   
               
               
                   
                 substrate (Y4F) 
                   
                   
               
               
                 pB2H S1.2pro   
                 An early version of B2H that (i)  
                 K 
                 Fox Lab 
               
               
                   
                 lacks PTP1B, (ii) contains 
                   
                   
               
               
                   
                 LuxAB, and (iii) includes the  
                   
                   
               
               
                   
                 pro RBS for Src. 
                   
                   
               
               
                 pB2H S1.2pro.mut   
                 Identical to pB2H S1.2pro  except  
                 K 
                 Fox Lab 
               
               
                   
                 for a mutation in the 
                   
                   
               
               
                   
                 substrate (Y4F) 
                   
                   
               
               
                 pB2H S1.2Sal28   
                 An early version of B2H that (i)  
                 K 
                 Fox Lab 
               
               
                   
                 lacks PTP1B, (ii) contains 
                   
                   
               
               
                   
                 LuxAB, and (iii) includes the  
                   
                   
               
               
                   
                 Sal28 RBS for Src. 
                   
                   
               
               
                 pB2H S1.2Sal28.   
                 Identical to pB2H S1.2Sal28   
                 K 
                 Fox Lab 
               
               
                 
                   mut 
                 
                 except for a mutation in the 
                   
                   
               
               
                   
                 substrate (Y4F) 
                   
                   
               
               
                 pB2H S1.3RBS30   
                 An early version of B2H that (i)  
                 K 
                 Fox Lab 
               
               
                   
                 contains LuxAB and (ii) 
                   
                   
               
               
                   
                 includes the bb030 RBS  
                   
                   
               
               
                   
                 for PTP1B. 
                   
                   
               
               
                 pB2Hs S1.3RBS30.   
                 Identical to pB2H S1.3RBS30   
                 K 
                 Fox Lab 
               
               
                 
                   mut 
                 
                 except for a mutation in the 
                   
                   
               
               
                   
                 substrate (Y4F) 
                   
                   
               
               
                 pB2H S1.3RBS34   
                 An early version of B2H that (i)  
                 K 
                 Fox Lab 
               
               
                   
                 contains LuxAB and (ii) 
                   
                   
               
               
                   
                 includes the bb034 RBS for  
                   
                   
               
               
                   
                 PTP1B. 
                   
                   
               
               
                 pB2H S1.3RBS34.   
                 Identical to pB2H S1.3RBS34   
                 K 
                 Fox Lab 
               
               
                 
                   mut 
                 
                 except for a mutation in the 
                   
                   
               
               
                   
                 substrate (Y4F) 
                   
                   
               
               
                 pB2H S2RBS30   
                 An early version of B2H that (i)  
                 K 
                 Fox Lab 
               
               
                   
                 contains SpecR and (ii) 
                   
                   
               
               
                   
                 includes the bb030 RBS for  
                   
                   
               
               
                   
                 PTP1B. 
                   
                   
               
               
                 pB2H S2RBS30.   
                 Identical to pB2H S2RBS30    
                 K 
                 Fox Lab 
               
               
                 
                   mut 
                 
                 except for an inactivating 
                   
                   
               
               
                   
                 mutation in PTP1B (C215S) 
                   
                   
               
               
                 pB2H opt   
                 Final, optimized B2H that (i)  
                 K 
                 AG:  
               
               
                   
                 contains SpecR and (ii) 
                   
                 163830 
               
               
                   
                 includes the bb034 RBS  
                   
                   
               
               
                   
                 for PTP1B. 
                   
                   
               
               
                 pBH opt*   
                 Identical to pB2H opt  except for  
                 K 
                 AG:  
               
               
                   
                 an inactivating mutation in 
                   
                 163831 
               
               
                   
                 PTP1B (C215S) 
                   
                   
               
               
                 pB2H optX   
                 Identical to pB2H opt  except for a  
                 K 
                 AG:  
               
               
                   
                 mutation in the substrate 
                   
                 163832 
               
               
                   
                 domain (Y4F) 
                   
                   
               
               
                 pB2H 2   
                 Identical to pB2H opt  with TC- 
                 K 
                 AG:  
               
               
                   
                 PTP in place of PTP1B 
                   
                 163833 
               
               
                 pB2H 2 * 
                 Identical to pB2H 2  except for an  
                 K 
                 AG:  
               
               
                   
                 inactivating mutation in 
                   
                 163834 
               
               
                   
                 TC-PTP (R222M) 
                   
                   
               
               
                 pB2H 6   
                 Identical to pB2H opt  with SHP1  
                 K 
                 AG:  
               
               
                   
                 (catalytic domain) in place 
                   
                 163835 
               
               
                   
                 of PTP1B 
                   
                   
               
               
                 pB2H 6 * 
                 Identical to pB2H 6  except for an  
                 K 
                 AG:  
               
               
                   
                 inactivating mutation in 
                   
                 163836 
               
               
                   
                 SHP1 (R459M) 
                   
                   
               
               
                 pB2H 12   
                 Identical to pB2H opt  with  
                 K 
                 AG:  
               
               
                   
                 PTPN12 in place of PTP1B 
                   
                 163837 
               
               
                 pB2H 12 * 
                 Identical to pB2H 12  except for  
                 K 
                 AG:  
               
               
                   
                 an inactivating mutation in 
                   
                 163838 
               
               
                   
                 PTPN12 (Y64A) 
                   
                   
               
               
                 pMBIS 
                 A plasmid that harbors genes  
                 T 
                 AG:  
               
               
                   
                 for the mevalonate- 
                   
                 17817 
               
               
                   
                 dependent isoprenoid pathway  
                   
                   
               
               
                   
                 from  S. cerevisiae  and 
                   
                   
               
               
                   
                 harbors a tetracycline  
                   
                   
               
               
                   
                 resistance marker. 
                   
                   
               
               
                 pMBIS CmR   
                 A plasmid that harbors genes  
                 P 
                 Fox Lab 
               
               
                   
                 for the mevalonate- 
                   
                   
               
               
                   
                 dependent isoprenoid pathway  
                   
                   
               
               
                   
                 from  S. cerevisiae  and 
                   
                   
               
               
                   
                 harbors a chloramphenicol  
                   
                   
               
               
                   
                 resistance marker. 
                   
                   
               
               
                 pTrc99t 
                 A pTrc99a variant with BsaI 
                 C 
                 Fox Lab 
               
               
                   
                 removed for use in Golden 
                   
                   
               
               
                   
                 Gate cloning 
                   
                   
               
               
                 PTS ADS   
                 A plasmid that harbors ADS. 
                 C 
                 AG:  
               
               
                   
                   
                   
                 19040 
               
               
                 pTS ADS(D299A)   
                 A plasmid that harbors ADS  
                 C 
                 Fox Lab 
               
               
                   
                 (D299A, inactivating). 
                   
                   
               
               
                 pTS GHS   
                 A plasmid that harbors GHS. 
                 C 
                 AG:  
               
               
                   
                   
                   
                 19003 
               
               
                 pTS GHS(D343A)   
                 A plasmid that harbors GHS  
                 C 
                 Fox Lab 
               
               
                   
                 (D343A, inactivating). 
                   
                   
               
               
                 pTS ABA   
                 A plasmid that harbors ABA. 
                 C 
                 Fox Lab 
               
               
                 pTS ABA(D566A)   
                 A plasmid that harbors ABA  
                 C 
                 Fox Lab 
               
               
                   
                 (D566A, inactivating). 
                   
                   
               
               
                 pTS ABS   
                 A plasmid that harbors ABS  
                 C 
                 AG:  
               
               
                   
                 and GGPPS. 
                   
                 163840 
               
               
                 pTS ABS(D404A/   
                 A plasmid that harbors ABS  
                 C 
                 Fox Lab 
               
               
                 
                   D621A) 
                 
                 (D404A/D621A, inactivating) 
                   
                   
               
               
                   
                 and GGPPS. 
                   
                   
               
               
                 pTS TXS   
                 A plasmid that harbors TXS  
                 C 
                 AG:  
               
               
                   
                 and GGPPS. 
                   
                 163839 
               
               
                 pTS A0A166A5J3   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 A0A166A5J3 (Clade 1) 
                   
                   
               
               
                 pTS A0A0D9X4S7   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 A0A0D9X487 (Clade 1) 
                   
                   
               
               
                 pTS F2DRF1   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 F2DRF1 (Clade 1) 
                   
                   
               
               
                 pTS A2XI80   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 A2XI80 (Clade 2) 
                   
                   
               
               
                 pTS A0AOD9ZGD1   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 A0A0D9ZGD1 (Clade 2) 
                   
                   
               
               
                 pTS A0A0K9RZT8   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 A0A0K9RZT8 (Clade 2) 
                   
                   
               
               
                 pTS A0A1I1AC30   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 A0A1I1AC30 (Clade 3) 
                   
                   
               
               
                 pTS A0A1S3XW43   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 A0A1S3XW43 (Clade 3) 
                   
                   
               
               
                 pTS A0A0D3D8G7   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 A0A0D3D8G7 (Clade 3) 
                   
                   
               
               
                 pTS B9IF04   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 B9IF04 (Clade 4) 
                   
                   
               
               
                 pTS A0A067L3D3   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 A0A067L3D3 (Clade 4) 
                   
                   
               
               
                 pTS A0A0C2TFL3   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 A0A0C2TFL3 (Clade 4) 
                   
                   
               
               
                 pTS A0A022S1C8   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 A0A022S1C8 (Clade 5) 
                   
                   
               
               
                 pTS G4TNA6   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 G4TNA6 (Clade 5) 
                   
                   
               
               
                 pTS A0A1L7WMZ8   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 A0A1L7WMZ8 (Clade 5) 
                   
                   
               
               
                 pTS A0A078IZJ5   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 A0A078IZJ5 (Clade 6) 
                   
                   
               
               
                 pTS A0A0C9VSL7   
                 A plasmid that harbors  
                 C 
                 AG:  
               
               
                   
                 A0A0C9VSL7 (Clade 6) 
                   
                 163841 
               
               
                 pTS G2QRS0   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 G2QRS0 (Clade 6) 
                   
                   
               
               
                 pTS A0A2H3DKU3   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 A0A2H3DKU3 (Clade 7) 
                   
                   
               
               
                 pTS A0A0D2L718   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 A0A0D2L718 (Clade 7) 
                   
                   
               
               
                 pTS S9Q922   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 S9Q922 (Clade 7) 
                   
                   
               
               
                 pTS T1LTV1   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 T1LTV1 (Clade 8) 
                   
                   
               
               
                 pTS A0A2S7XU99   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 A0A287XU99 (Clade 8) 
                   
                   
               
               
                 pTSA 0A0G2ZSL3   
                 A plasmid that harbors  
                 C 
                 Fox Lab 
               
               
                   
                 A0A0G2ZSL3 (Clade 8) 
                   
                   
               
               
                 pET21b ptp1b   
                 A plasmid that encodes a His- 
                 C 
                 N/A +   
               
               
                   
                 tagged catalytic domain of 
                   
                   
               
               
                   
                 PTP1B (for protein expression) 
                   
                   
               
               
                 pET16B TCPTP   
                 A plasmid that encodes a His- 
                 C 
                 Fox Lab 
               
               
                   
                 tagged catalytic domain of 
                   
                   
               
               
                   
                 TCPTP (for protein expression) 
               
               
                   
               
               
                 *Antibiotic resistance: carbenicillin (C, 50 μg/ml), kanamycin (K, 50 μg/ml), tetracycline (T, 10 μg/ml), chloramphenicol (P, 34 μg/ml), and spectinomycin (S, conditional). 
               
               
                   + This plasmid was a kind gift from Nicholas Tonks of Cold Spring Harbor Laboratory. 
               
               
                   *   * AG = Addgene accession # (Addgene.com). 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Primers used to assemble pathways for terpenoid biosynthesis. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 F Primer 
                   
                 R Primer 
                   
               
               
                 Component 
                 SEQ ID NO: 
                 F Primer 
                 SEQ ID NO: 
                 R Primer 
               
               
                   
               
               
                 GGPPS into 
                  76 
                 TATTGAGCTCCACCGCGGA 
                  80 
                 TATTGTCGACTTATTTATTAC 
               
               
                 pTrc99t 
                   
                 GGAGGAATG 
                   
                 GCTGGATGATGTAGTC 
               
               
                   
               
               
                 TXS into 
                  77 
                 TATTGGTCTCCCATGAGCA 
                  81 
                 TATTGGTCTCCGTCCTTCCAA 
               
               
                 pTrc99t 
                   
                 GCAGCACTGGCAC 
                   
                 CGCATTCAACATGTTG 
               
               
                   
               
               
                 ABS into 
                  78 
                 ATAAAGGTCTCCCATGGTG 
                  82 
                 TATTAGGTCTCGAGCTCTTAG 
               
               
                 pTrc99t 
                   
                 AAACGAGAATTTCCTCCAG 
                   
                 GCAACTGGTTGGAAGAGGC 
               
               
                   
               
               
                 pMBIS TetR- 
                  79 
                 AGATCACTACCGGGCGTAT 
                  83 
                 GCCGCCGGCTTCCATTTATTA 
               
               
                 &gt;CmR 
                   
                 TTTTTGAGTTATCGAGATT 
                   
                 CGCCCCGCCCTG 
               
               
                   
                   
                 TTCAGGAGCTAAGGAAGCT 
                   
                   
               
               
                   
                   
                 AAAATGGAGAAAAAAATCA 
                   
                   
               
               
                   
                   
                 CTGGATATACCAC 
                   
                   
               
               
                   
               
               
                 ABA into 
                 130 
                 AACAATTTCACACAGGAAA 
                 131 
                 GCCTGCAGGTCGACTCTAGAT 
               
               
                 pTrc99 
                   
                 CAGACCATGGCGGGTGTTT 
                   
                 TACAGCGGCAGCGGTTC 
               
               
                   
                   
                 CTGCG 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Primers used for site-directed mutagenesis. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 F Primer 
                   
                 R Primer 
                   
               
               
                 Mutant 
                 SEQ ID NO: 
                 F Primer 
                 SEQ ID NO: 
                 R Primer 
               
               
                   
               
               
                 PTP1B 
                  84 
                 GTCCAGTACTTTATTGGGGTT 
                 107 
                 ATCTCGGACATGCTCAGTTCCA 
               
               
                 (C215S) 
                   
                 CAGGCGGATGGAACTGAGCAT 
                   
                 TCCGCCTGAACCCCAATAAAGT 
               
               
                   
                   
                 GTCCGAGAT 
                   
                 ACTGGAC 
               
               
                   
               
               
                 TCPTP 
                 132 
                 CAGAGAGAAGGTGCCAGACAT 
                 136 
                 TGTAGTGCAGGCATTGGGATGT 
               
               
                 (R222M) 
                   
                 CCCAATGCCTGCACTACA 
                   
                 CTGGCACCTTCTCTCTG 
               
               
                   
               
               
                 SHP1 
                 133 
                 CAATGATGGTGCCTGTCATGC 
                 137 
                 CAGCGCCGGCATCGGCATGACA 
               
               
                 (R459M) 
                   
                 CGATGCCGGCGCTG 
                   
                 GGCACCATCATTG 
               
               
                   
               
               
                 PTPN12 
                 134 
                 GCTGTGATCAAATGGCAGTAT 
                 138 
                 GAAAAAGAAGAAAATGTTAAAA 
               
               
                 (Y64A) 
                   
                 GTCCTTCGCTCTGTTCTTTTT 
                   
                 AGAACAGAGCGAAGGACATACT 
               
               
                   
                   
                 AACATTTTCTTCTTTTTC 
                   
                 GCCATTTGATCACAGC 
               
               
                   
               
               
                 ABS 
                 85 
                 GAGAGAGAATCCTGTTCCTGA 
                 108 
                 GAAGGCCCATGGCTGTATCCGC 
               
               
                 (D404A) 
                   
                 TATTGCGGATACAGCCATGGG 
                   
                 AATATCAGGAACAGGATTCTCT 
               
               
                   
                   
                 CCTTC 
                   
                 CTC 
               
               
                   
               
               
                 ABS 
                 86 
                 ACAAAAACTTCCAATTTCACT 
                 109 
                 CCATGGGCGTCATAAAGATCCG 
               
               
                 (D621A) 
                   
                 GTTATTTTAGCGGATCTTTAT 
                   
                 CTAAAATAACAGTGAAATTGGA 
               
               
                   
                   
                 GACGCCCATGG 
                   
                 AGTTTTTGT 
               
               
                   
               
               
                 ADS 
                 87 
                 CGTAAGCATCGTAAGTGTCCG 
                 110 
                 GCTGTTATCACCCTGATCGCGG 
               
               
                 (D299A) 
                   
                 CGATCAGGGTGATAACAGC 
                   
                 ACACTTACGATGCTTACG 
               
               
                   
               
               
                 GHS 
                 88 
                 CCCATGCGTGTCGTATAAGTC 
                 111 
                 CGATCTTGATGACAATGTTAGC 
               
               
                 (D343A) 
                   
                 CGCTAACATTGTCATCAAGAT 
                   
                 GGACTTATACGACACGCATGGG 
               
               
                   
                   
                 CG 
                   
                   
               
               
                   
               
               
                 MidT 
                 100 
                 CAGCTGCGGAACCGCAGTTTG 
                 123 
                 ATCGGAATTTCTTCAAACTGCG 
               
               
                 Substrate 
                   
                 AAGAAATTCCGAT 
                   
                 GTTCCGCAGCTG 
               
               
                 (Y/F) 
                   
                   
                   
                   
               
               
                   
               
               
                 p130Cas 
                 101 
                 TGGATGGAGGACTTTGACTTC 
                 124 
                 GTCAAAGTCCTCCATCCACGCA 
               
               
                 Substrate 
                   
                 GTCCACCTACAGGGGTAATAA 
                   
                 GCTGCACGACG 
               
               
                 (Y/F) 
                   
                 CAATTC 
                   
                   
               
               
                   
               
               
                 SH2 
                 102 
                 CTCTCCGTTTCTGACTTTGAC 
                 125 
                 AAGTCAGAAACGGAGAGGGCAT 
               
               
                 (Superbinder 
                   
                 AACGCCAAGGGGCTCAATGTG 
                   
                 AGGCACCTTTTACCGTCTCGCT 
               
               
                 mutations) 
                   
                 CTGCACTACAAGATCCGCAAG 
                   
                 CTCCCG 
               
               
                   
                   
                 CTG 
                   
                   
               
               
                   
               
               
                 SH2 (L13K 
                 103 
                 AAACACTACCTGATCCGCAAG 
                 126 
                 GCTGTCCAGCTTGCGGATCAGG 
               
               
                 K15L)* 
                   
                 CTGGACAGC 
                   
                 TAGTGTTTCACATTGAGCCCCT 
               
               
                   
                   
                   
                   
                 TGGC* 
               
               
                   
               
               
                 pTrc99a 
                 104 
                 TATTGGTCTCTCGCGGTATCA 
                 127 
                 TATTGGTCTCAGTGACCCCACA 
               
               
                 (remove BsaI 
                   
                 TTGCAGCAC 
                   
                 CTACCATCGG 
               
               
                 sites) piece 1 
                   
                   
                   
                   
               
               
                   
               
               
                 pTrc99a 
                 105 
                 TATTGGTCTCATCACCCCATG 
                 128 
                 TATTGGTCTCACGCGTGACCCA 
               
               
                 (remove BsaI 
                   
                 CGAGAGTAGG 
                   
                 CGCTCACCG 
               
               
                 sites) piece 2 
                   
                   
                   
                   
               
               
                   
               
               
                 ABA D/A 
                 135 
                 AGGTGTCGTACATGTCCGCCA 
                 139 
                 CTGCAGACCGTTCTGGCGGACA 
               
               
                   
                   
                 GAACGGTCTGCAG 
                   
                 TGTACGACACCT 
               
               
                   
               
               
                 *The original superbinder primer mutated the incorrect lysine residue (13 vs. 15). This 
               
               
                 primer corrects that error. The residue numbering system used for this protein matches that 
               
               
                 of Kaneko et. al. 20   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11a 
               
             
            
               
                   
               
               
                 Scaling factor for amorphadiene/caryophyllene (m/z = 204) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Technical 
                 A std    
                 A ref    
                 C std   
                 C ref    
                   
               
               
                 Replicate 
                 (counts*min) 
                 (counts*min) 
                 (μg/mL) 
                 (μg/mL) 
                 R 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 1 
                 74520 
                 88358 
                 20 
                 0.4 
                 0.017 
               
               
                 2 
                 71037 
                 142415 
                 20 
                 0.4 
                 0.010 
               
               
                 3 
                 75761 
                 49011 
                 20 
                 0.4 
                 0.031 
               
               
                   
                   
                   
                   
                 Avg R 
                 0.019 (0.006) 
               
               
                   
               
               
                 *R was computed using eq. 2. Standard error is shown in parentheses. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11b 
               
             
            
               
                   
               
               
                 Scaling factor for taxadiene/caryophyllene (m/z = 93) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Technical 
                 A std    
                 A ref    
                 C std   
                 C ref    
                   
               
               
                 Replicate 
                 (counts*min) 
                 (counts*min) 
                 (μg/mL) 
                 (μg/mL) 
                 R 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 1 
                 1399872 
                 847009 
                 20 
                 10 
                 0.83 
               
               
                 2 
                 1247250 
                 605265 
                 20 
                 10 
                 1.0 
               
               
                 3 
                 1291028 
                 547740 
                 20 
                 10 
                 1.2 
               
               
                   
                   
                   
                   
                 Avg R 
                 1.0 (0.10) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11c 
               
             
            
               
                   
               
               
                 Scaling factor for amorphadiene/methyl abietate (m/z = 121) 
               
            
           
           
               
               
               
               
               
               
            
               
                 Technical 
                 A std    
                 A ref   
                 C std   
                 C ref   
                   
               
               
                 Replicate 
                 (counts * min) 
                 (counts * min) 
                 (μg/mL) 
                 (μg/mL) 
                 R 
               
               
                   
               
               
                 1 
                 949492 
                  868168 
                 20 
                 3.162 
                 0.17 
               
               
                 2 
                 920694 
                  908257 
                 20 
                 3.162 
                 0.16 
               
               
                 3 
                 898594 
                 1106474 
                 20 
                 3.162 
                 0.13 
               
               
                   
                   
                   
                   
                 Avg R 
                 0.15 
               
               
                   
                   
                   
                   
                   
                 (0.01) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12a 
               
             
            
               
                   
               
               
                 Analysis of the inhibition of PTP1B 1-321  by amorphadiene. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SSE 
                   
                   
                   
                 Fit par. 
               
               
                 Model 
                 (μM 2 /s 2 ) 
                 DF 
                 Criteria 
                 Reference 
                 (μM) 
               
               
                   
               
               
                 Competitive 
                 0.14 
                 27 
                 Δ i  = 51.2 
                 noncompetitive 
                 K i  = 2.85 
               
               
                 Uncompetitive** 
                 0.023 
                 27 
                 Δ i  = 1.16 
                 noncompetitive 
                 K i  = 46.3 
               
               
                 Noncompetitive** 
                 0.023 
                 27 
                   
                   
                 K i  = 52.6* 
               
               
                 Mixed 
                 0.022 
                 26 
                 F = 0.47 
                 noncompetitive 
                 K i,c  = 86.2 
               
               
                   
                   
                   
                 p = 0.972 
                   
                 K i,u  = 50.1 
               
               
                   
               
               
                 *The SSEs of the uncompetitive and noncompetitive models are indistinguishable from one another. 
               
               
                 **Indicate models of best fit. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12b 
               
             
            
               
                   
               
               
                 Analysis of the inhibition of PTP1B 1-321  by α-bisabolene. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SSE 
                   
                   
                   
                 Fit par. 
               
               
                 Model 
                 (μM 2 /s 2 ) 
                 DF 
                 Criteria 
                 Reference 
                 (μM)) 
               
               
                   
               
               
                 Competitive 
                 0.082 
                 27 
                 Δ i  = 39.1 
                 noncompetitive 
                 K i  = 1.05 
               
               
                 Uncompetitive** 
                 0.023 
                 27 
                 Δ i  = 3.81 
                 noncompetitive 
                 K i  = 11.7 
               
               
                 Noncompetitive** 
                 0.021 
                 27 
                   
                   
                 K i  = 13.1 
               
               
                 Mixed 
                 0.020 
                 26 
                 F = 0.24 
                 noncompetitive 
                 K i,c  = 9.51 
               
               
                   
                   
                   
                 p = 1.0 
                   
                 K i,u  = 13.7 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12c 
               
             
            
               
                   
               
               
                 Analysis of the inhibition of PTP1B 1-321  by alpha bisabolol. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SSE 
                   
                   
                   
                 Fit par. 
               
               
                 Model 
                 (μM 2 /s 2 ) 
                 DF 
                 Criteria 
                 Reference 
                 (μM) 
               
               
                   
               
               
                 Competitive 
                 0.039 
                 27 
                 Δ i  = 34.4 
                 uncompetitive 
                 K i  = 178 
               
               
                 Uncompetitive** 
                 0.011 
                 27 
                   
                   
                 K i  = 469 
               
               
                 Noncompetitive** 
                 0.013 
                 27 
                 Δ i  = 4.65 
                 uncompetitive 
                 K i  = 541 
               
               
                 Mixed 
                 0.011 
                 26 
                 F = 0 
                 uncompetitive 
                 K i,c  = 
               
               
                   
                   
                   
                   
                   
                 3.5e 16   
               
               
                   
                   
                   
                 p = 1.0 
                   
                 K i,u  = 469 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12d 
               
             
            
               
                   
               
               
                 Analysis of the inhibition of PTP1B 1-321  by dihydroartimesnic acid. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SSE 
                   
                   
                   
                 Fit par. 
               
               
                 Model 
                 (μM 2 /s 2 ) 
                 DF 
                 Criteria 
                 Reference 
                 (μM) 
               
               
                   
               
               
                 Competitive 
                 0.129 
                 21 
                 Δ i  = 60.7 
                 noncompetitive 
                 K i  = 178 
               
               
                 Uncompetitive 
                 0.025 
                 27 
                 Δ i  = 15.2 
                 noncompetitive 
                 K i  = 469 
               
               
                 Noncompetitive 
                 0.015 
                 27 
                   
                   
                 K i  = 541 
               
               
                 Mixed** 
                 0.013 
                 26 
                 F = 2.69 
                 noncompetitive 
                 K i,c  = 3.5e 16   
               
               
                   
                   
                   
                 p = 6.9e −3   
                   
                 K i,u  = 469 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12e 
               
             
            
               
                   
               
               
                 Analysis of the inhibition of TCPTP 1-317  by amorphadiene. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SSE 
                   
                   
                   
                 Fit par. 
               
               
                 Model 
                 (μM 2 /s 2 ) 
                 DF 
                 Criteria 
                 Reference 
                 (μM) 
               
               
                   
               
               
                 Competitive 
                 0.053 
                 27 
                 Δ i  = 41.1 
                 uncompetitive 
                 K i  = 87.2 
               
               
                 Uncompetitive** 
                 0.012 
                 27 
                   
                   
                 K i  = 356 
               
               
                 Noncompetitive** 
                 0.013 
                 27 
                 Δ i  = 2.22 
                 uncompetitive 
                 K i  = 400 
               
               
                 Mixed 
                 0.012 
                 26 
                 F = 0 
                 uncompetitive 
                 K i,c  = 
               
               
                   
                   
                   
                   
                   
                 3.7e 15   
               
               
                   
                   
                   
                 p = 1.0 
                   
                 K i,u  = 356 
               
               
                   
               
               
                 *The SSEs of the uncompetitive and noncompetitive models are indistinguishable from one another. 
               
               
                 **Indicate models of best fit. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12f 
               
             
            
               
                   
               
               
                 Analysis of the inhibition of TCPTP 1-317  by α-bisabolene. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SSE 
                   
                   
                   
                 Fit par. 
               
               
                 Model 
                 (μM 2 /s 2 ) 
                 DF 
                 Criteria 
                 Reference 
                 (μM) 
               
               
                   
               
               
                 Competitive 
                 0.046 
                 27 
                 Δ i  = 37.6 
                 uncompetitive 
                 K i  = 13.7 
               
               
                 Uncompetitive** 
                 0.012 
                 27 
                   
                   
                 K i  = 69.2 
               
               
                 Noncompetitive** 
                 0.012 
                 27 
                 Δ i  = 1.12 
                 uncompetitive 
                 K i  = 76.2 
               
               
                 Mixed 
                 0.012 
                 26 
                 F = 0 
                 uncompetitive 
                 K i,c  = 3610 
               
               
                   
                   
                   
                 p = 1.0 
                   
                 K i,u  = 69.3 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12g 
               
             
            
               
                   
               
               
                 Analysis of the inhibition of PTP1B 1-281  by amorphadiene. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SSE 
                   
                   
                   
                 Fit par. 
               
               
                 Model 
                 (μM 2 /s 2 ) 
                 DF 
                 Criteria 
                 Reference 
                 (μM) 
               
               
                   
               
               
                 Competitive 
                 0.010 
                 27 
                 Δ i  = 16.3 
                 noncompetitive 
                 K i  = 37.9 
               
               
                 Uncompetitive** 
                 0.006 
                 27 
                 Δ i  = 3.51 
                 noncompetitive 
                 K i  = 210 
               
               
                 Noncompetitive** 
                 0.006 
                 27 
                   
                   
                 K i  = 244 
               
               
                 Mixed 
                 0.006 
                 26 
                 F = 0.41 
                 noncompetitive 
                 K i,c  = 157 
               
               
                   
                   
                   
                 p = 0.99 
                   
                 K i,u  = 271 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12h 
               
             
            
               
                   
               
               
                 Analysis of the inhibition of PTP1B 1-281  by α-bisabolene. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SSE 
                   
                   
                   
                 Fit par. 
               
               
                 Model 
                 (μM 2 /s 2 ) 
                 DF 
                 Criteria 
                 Reference 
                 (μM) 
               
               
                   
               
               
                 Competitive 
                 0.012 
                 27 
                 Δ i  = 14.4 
                 noncompetitive 
                 K i  = 6.51 
               
               
                 Uncompetitive** 
                 0.008 
                 27 
                 Δ i  = 1.41 
                 noncompetitive 
                 K i  = 40.0 
               
               
                 Noncompetitive** 
                 0.007 
                 27 
                   
                   
                 K i  = 46.3 
               
               
                 Mixed 
                 0.007 
                 26 
                 F = 0 
                 noncompetitive 
                 K i,c  = 39.0 
               
               
                   
                   
                   
                 p = 1.0 
                   
                 K i,u  = 47.7 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12i 
               
             
            
               
                   
               
               
                 Analysis of the inhibition of TCPTP 1-281  by amorphadiene. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SSE 
                   
                   
                   
                 Fit par. 
               
               
                 Model 
                 (μM 2 /s 2 ) 
                 DF 
                 Criteria 
                 Reference 
                 (μM) 
               
               
                   
               
               
                 Competitive 
                 0.005 
                 27 
                 Δ i  = 22.9 
                 uncompetitive 
                 K i  = 87.2 
               
               
                 Uncompetitive** 
                 0.002 
                 27 
                   
                   
                 K i  = 356 
               
               
                 Noncompetitive** 
                 0.002 
                 27 
                 Δ i  = 0.83 
                 uncompetitive 
                 K i  = 400 
               
               
                 Mixed 
                 0.002 
                 26 
                 F = 0.03 
                 uncompetitive 
                 K i,c  = 
               
               
                   
                   
                   
                   
                   
                 3.7e 15   
               
               
                   
                   
                   
                 p = 1.0 
                   
                 K i,u  = 356 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12j 
               
             
            
               
                   
               
               
                 Analysis of the inhibition of TCPTP 1-281  by α-bisabolene. 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SSE 
                   
                   
                   
                 Fit par. 
               
               
                 Model 
                 (μM 2 /s 2 ) 
                 DF 
                 Criteria 
                 Reference 
                 (μM) 
               
               
                   
               
               
                 Competitive 
                 0.083 
                 27 
                 Δ i  = 39.1 
                 noncompetitive 
                 K i  = 13.7 
               
               
                 Uncompetitive** 
                 0.023 
                 27 
                 Δ i  = 3.81 
                 noncompetitive 
                 K i  = 69.2 
               
               
                 Noncompetitive** 
                 0.021 
                 27 
                   
                   
                 K i  = 76.2 
               
               
                 Mixed 
                 0.020 
                 26 
                 F = 0 
                 noncompetitive 
                 K i,c  = 3610 
               
               
                   
                   
                   
                 p = 1.0 
                   
                 K i,u  = 69.3 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12k 
               
             
            
               
                   
               
               
                 Analysis of the inhibition of PTP1B 1-321  by (+)1-(10),4-cadinadiene 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SSE 
                   
                   
                   
                 Fit par. 
               
               
                 Model 
                 (μM 2 /s 2 ) 
                 DF 
                 Criteria 
                 Reference 
                 (μM) 
               
               
                   
               
               
                 Competitive 
                 0.115 
                 27 
                 Δ i  = 48.3 
                 uncompetitive 
                 K i  = 14.75 
               
               
                 Uncompetitive** 
                 0.020 
                 27 
                   
                   
                 K i  = 168.09 
               
               
                 Noncompetitive** 
                 0.022 
                 27 
                 Δ i  = 2.5 
                 uncompetitive 
                 K i  = 190.44 
               
               
                 Mixed 
                 0.020 
                 26 
                 F = 0 
                 uncompetitive 
                 K i,c  =  
               
               
                   
                   
                   
                   
                   
                 5689.38 
               
               
                   
                   
                   
                 p = 1.0 
                   
                 K i,u  = 
               
               
                   
                   
                   
                   
                   
                 168.78 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12l 
               
             
            
               
                   
               
               
                 Analysis of the inhibition of SHP2 223-565  by Amorphadiene 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 SSE 
                   
                   
                   
                 Fit par. 
               
               
                 Model 
                 (μM 2 /s 2 ) 
                 DF 
                 Criteria 
                 Reference 
                 (μM) 
               
               
                   
               
               
                 Competitive 
                  .0024 
                 27 
                 Δ i  = 10.6 
                 noncompetitive 
                 K i  = 25.1 
               
               
                 Uncompetitive** 
                  .0017 
                 27 
                 Δ i  = 0.5 
                 noncompetitive 
                 K i  = 116.51 
               
               
                 Noncompetitive** 
                  .0017 
                 27 
                   
                   
                 K i  = 145.69 
               
               
                 Mixed 
                 0.0017 
                 26 
                 F = 0.15 
                 noncompetitive 
                 K i,c  = 
               
               
                   
                   
                   
                   
                   
                 236.21 
               
               
                   
                   
                   
                 p = 1.0 
                   
                 K i,u  = 
               
               
                   
                   
                   
                   
                   
                 132.37 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Data collection and refinement statistics (molecular replacement) 
               
            
           
           
               
               
               
            
               
                   
                 PTP1B: amorphadiene 
                 PTP1B: α-bisabolol 
               
               
                   
                 (6W30) 
                 (N/A***) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 Data collection 
                   
                   
               
               
                 Space group 
                   
                   
               
               
                 Cell dimensions 
                   
                   
               
               
                 a, b, c (Å) 
                 89.03, 89.03, 105.56 
                 89.28, 89.28, 105.51 
               
               
                 α, β, γ (°) 
                 90.00, 90.00, 120.00 
                 90.00, 90.00, 120.00 
               
               
                 Resolution (Å) 
                 62.26-2.10 (2.13-2.10)* 
                 77.32-2.11 (2.15-2.11) 
               
               
                 R sym  or R merge   
                 0.130 (0.442) 
                 0.086 (0.331) 
               
               
                 I/σI 
                 5.4 (1.0) 
                 6.7 (1.1) 
               
               
                 Completeness (%) 
                 99.8 (93.3) 
                 100.0 (98.5)  
               
               
                 Redundancy 
                 10.7 (10.8) 
                 12.1 (12.3) 
               
               
                 Refinement 
                   
                   
               
               
                 Resolution (Å) 
                 44.52-2.10 (2.17-2.10) 
                 62.37-2.11 (2.18-2.11) 
               
               
                 No. reflections 
                 28,654 
                 28,479 
               
               
                 R work /R free   
                 0.20/0.24 
                 0.19/0.24 
               
               
                 No. atoms 
                   
                   
               
               
                 Protein 
                 2355 
                 2320 
               
               
                 Ligand/ion 
                 22 
                 17 
               
               
                 Water 
                 170 
                 270 
               
               
                 B-factors 
                   
                   
               
               
                 Protein 
                 37 
                 30 
               
               
                 Ligand/ion 
                 90/61 
                 66/37 
               
               
                 Water 
                 47 
                 43 
               
               
                 R.m.s. deviations 
                   
                   
               
               
                 Bond lengths (Å) 
                 0.42 
                 0.42 
               
               
                 Bond angles (°) 
                 0.56 
                 0.54 
               
               
                   
               
               
                 *Values in parentheses correspond to the highest-resolution shell. 
               
               
                 **Number of crystals used for each structure: 1 
               
               
                 ***In light of the results detailed in FIG. 31, we elected not to deposit this structure into the protein data bank. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Details of hypothesis testing 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 95% 
                   
               
               
                   
                 Null  
                   
                   
                   
                   
                 confidence 
                 P- 
               
               
                 FIG. 
                 hypothesis 
                 Δμ 
                 Test 
                 DF 
                 t 
                 intervals 
                 value 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 3h 
                 AD-(−) = 0 
                 0.212 
                 t-test, 
                 2 
                 6.61 
                 (0.092,  
                 0.02 
               
               
                   
                   
                   
                 unequal 
                   
                   
                 0.332) 
                   
               
               
                   
                   
                   
                 variance 
                   
                   
                   
                   
               
               
                 3h 
                 AB-(−) = 0 
                 0.310 
                 t-test, 
                 2 
                 13.5 
                 (0.138,  
                 0.005 
               
               
                   
                   
                   
                 unequal 
                   
                   
                 0.482) 
                   
               
               
                   
                   
                   
                 variance 
                   
                   
                   
                   
               
               
                 3h 
                 AD- 
                 0.124 
                 t-test, 
                 3 
                 3.59 
                 (0.069,  
                 0.04 
               
               
                   
                 DHA = 0 
                   
                 unequal 
                   
                   
                 0.179) 
                   
               
               
                   
                   
                   
                 variance 
                   
                   
                   
                   
               
               
                 3h 
                 AB- 
                 0.309 
                 t-test, 
                 3 
                 12.6 
                 (0.170,  
                 0.001 
               
               
                   
                 ABOL = 0 
                   
                 unequal 
                   
                   
                 0.447) 
                   
               
               
                   
                   
                   
                 variance 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Ligand efficiency. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 # Heavy 
                 Ligand Efficiency 
               
               
                   
                 Ligand 
                 IC 50  (μM) 
                 Atoms 
                 (kcal/mol-atom)* 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Amorphadiene 
                 50 
                 15 
                 0.39 
               
               
                   
                 α-bisabolene 
                 13 
                 15 
                 0.44 
               
               
                   
                 BBR 
                 8 
                 41 
                 0.17 
               
               
                   
                 MSI-1436 
                 0.6 
                 47 
                 0.17 
               
               
                   
                   
               
               
                   
                 *Ligand efficiency = (−2.303RT)/HAC * log(IC 50 ), where R is the gas constant, T is the temperature in K, and HAC is the number of heavy atoms. 
               
            
           
         
       
     
     OTHER EMBODIMENTS 
     All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. 
     From the above description, one skilled in the art can easily ascertain the essential characteristics of the present disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Thus, other embodiments are also within the claims. 
     EQUIVALENTS AND SCOPE 
     While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure. 
     All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms. 
     All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document. 
     The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” 
     The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. 
     As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and