Sensors for Aromatic Compounds and Methods of Making and Using Same
20220372585 · 2022-11-24
Assignee
Inventors
- Tae Seok Moon (St. Louis, MO, US)
- Hyojeong Yi (St. Louis, MO, US)
- Matthew Amrofell (St. Louis, MO, US)
- Chenggang Xi (St. Louis, MO, US)
- Austin Rottinghaus (St. Louis, MO, US)
Cpc classification
C12Q1/6897
CHEMISTRY; METALLURGY
International classification
C12Q1/6897
CHEMISTRY; METALLURGY
Abstract
Among the various aspects of the present disclosure is the provision of molecular sensors, microbial sensors, constructs, systems, and methods for selectively detecting aromatic compounds.
Claims
1. An engineered molecular sensor comprising: an engineered regulator protein or enzyme (e.g., TrpR, TyrR, TynA, FeaR) comprising a ligand-protein binding site; a reporter (comprising a signaling moiety, e.g., GFP); and a promoter (e.g., PtyrP) capable of inducing or repressing the reporter in the presence or absence of a target aromatic compound bound to the engineered molecular sensor, wherein, the engineered regulator protein or the ligand-protein binding site or a sequence encoding the engineered regulator protein or the ligand-protein binding site of the engineered regulator protein is at least about 80% identical to a WT regulator protein or a WT regulator ligand-protein binding site; or a sequence encoding the engineered regulator protein or ligand binding site thereof is at least about 80% identical to a sequence encoding the WT regulator protein or the WT regulator ligand-protein binding site; or functional fragment thereof, the engineered regulator protein having ligand binding activity; and wherein the engineered molecular sensor is optionally genomically integrated into a microorganism, optionally selected from a probiotic or is a purified cell-free sensor.
2. The engineered molecular sensor of claim 1, wherein the engineered regulator protein or enzyme is an engineered TrpR, TyrR, TynA, or FeaR protein.
3. The engineered molecular sensor of claim 1, wherein (a) the engineered regulator protein is an engineered TyrR and is an aromatic amino acid-specific sensor, wherein the aromatic amino acid-specific sensor is a phenylalanine (Phe)- or a tyrosine (Tyr)-specific sensor; (b) the engineered regulator protein or enzyme is an engineered FeaR or TynA and is an aromatic amine-specific sensor, wherein the aromatic amine-specific sensor is a dopamine (DA)-, phenylethylamine (PEA)-, tyramine (Tyra)-, or tryptamine (Trypta)-specific sensor; or (c) the engineered regulator protein is an engineered TrpR and is a tryptophan (Trp)-, 5-hydroxytryptophan (5-HTP)-, or tryptamine (Trpta)-specific sensor.
4. The engineered molecular sensor of claim 1, wherein the engineered regulator protein or enzyme is an engineered TrpR, TyrR, TynA, or FeaR, and wherein the engineered TrpR, TyrR, TynA, or FeaR comprises at least one mutation to a ligand-protein binding site having specific amino acid binding activity.
5. The engineered molecular sensor of claim 3, wherein: PEA induces reporter expression; Phe induces reporter expression; or Tyr represses or induces reporter expression.
6. The engineered molecular sensor of claim 3, wherein: carboxylic acids 3,4-dihydroxyphenylacetic acid (DOPAC), phenylacetic acid (PAA), 4-hydroxyphenylacetic acid (HPPA), or indole-3-acetic acid (IAA) do not induce reporter expression or wherein IAA induces reporter expression.
7. The engineered molecular sensor of claim 1, wherein the engineered regulator protein is an engineered TyrR and the engineered TyrR comprises one or more of the following mutations: E274Q+T14V mutations in TyrR inducing reporter expression in the presence of Phe; E274Q+D103S mutations in TyrR inducing reporter expression in the presence of Phe; or an R10F mutation in TyrR repressing reporter expression in the presence of Tyr, wherein TyrR protein sequence is at least about 80% identical to SEQ ID NO: 124 or TyrR ligand binding site is at least about 80% identical to residues 7-274 of SEQ ID NO: 124; a polypeptide encoded by SEQ ID NO: 11 or the WT ligand binding site sequence; or functional fragment or conservative substitution thereof of TyrR having ligand binding functional activity.
8. A TyrR-based selective or specific sensor specifically detecting phenylalanine (Phe) or tyrosine (Tyr) comprising: a TyrR or functional mutant or variant thereof; and a reporter gene (comprising a signaling moiety, e.g., GFP) operably linked to an inducible promoter (e.g., PtyrP promoter).
9. The TyrR-based sensor of claim 8, wherein the inducible promoter comprises an activating response to Phe or a repressing response to Tyr or both Phe and Tyr.
10. The TyrR-based sensor of claim 8, wherein TyrR is selected from: wild type (VVT) TyrR (SEQ ID NO: 124) or having at least about 80% identity to WT TyrR (SEQ ID NO: 124) and the reporter gene overexpresses if Phe is present; TyrR or TyrR having at least about 80% identity to TyrR comprising the mutation E274Q and the reporter gene overexpresses in the presence of Phe and Phe+Tyr; TyrR or TyrR having at least about 80% identity to TyrR comprising the mutation E274Q+T14V and the reporter gene overexpresses in the presence of Phe and Phe+Tyr and the reporter gene does not overexpress with Tyr in the absence of Phe; TyrR or TyrR having at least about 80% identity to TyrR comprising the mutation E274Q+D103S and the reporter gene overexpresses in the presence of Phe and Phe+Tyr and the reporter gene does not overexpress with Tyr in the absence of Phe; or TyrR or TyrR having at least about 80% identity to TyrR comprising the mutation R10F and the reporter gene is repressed with Tyr or Tyr+Phe and the reporter gene does not overexpress in the absence of Tyr.
11. The TyrR-based sensor of claim 8, wherein, the sensor is a target aromatic compound-inducible sensor or target aromatic compound-repressible sensor selected from: a Phe-inducible TyrR system (e.g., E274Q, E274Q+T14V, E274Q+D103H, E274Q+D103S), or Tyr-repressible TyrR system (e.g., R10F).
12. The TyrR-based sensor of claim 8, for use to kinetically diagnose or treat disorders that cause Phe-dysregulation without interference from intestinal Tyr.
13. The TyrR-based sensor of claim 8, comprising E274Q+T14V or E274Q+D103S variants of TyrR and wherein the TyrR-based sensor is sensitive to Phe in the presence or absence of Tyr and does not respond to Tyr alone.
14. The TyrR-based sensor of claim 8, comprising R10F variant of TyrR and wherein the TyrR-based sensor exhibits about a 12-fold repression in the presence of Tyr independent of the presence of Phe.
15. The TyrR-based sensor of claim 8, wherein the TyrR sensor has no significant response to Phe alone.
16. The TyrR-based sensor of claim 8, wherein a significant response is: under about 4000 au, under about 3000 au, or preferably at or under about 2000 au for Phe-inducible sensor; or above about 2000 au or preferably at or above 2000 au for a Phe-repressible sensor.
17. The engineered molecular sensor of claim 1, wherein the engineered regulator protein or enzyme comprises an engineered FeaR or TynA or both and the engineered FeaR or TynA comprises one or more of the following mutations: a G494S mutation in TynA inducing reporter expression in the presence of PEA; a G494S mutation in TynA and A81T mutation in FeaR (e.g., G494S*) inducing reporter expression in the presence of PEA; a G494S mutation in TynA and A81S mutation in FeaR inducing reporter expression in the presence of PEA; a A81T or A81S mutation in FeaR inducing reporter expression in the presence of PEA and Tyra; a A81T mutation in FeaR inducing reporter expression in the presence of PEA; an A81T mutation in FeaR and S414M mutation in TynA inducing reporter expression in the presence of PEA; an A81T mutation in FeaR and G415H mutation in TynA inducing reporter expression in the presence of PEA; an A81L, A81P, A81I, or A81N mutation in FeaR inducing reporter expression in the presence of PEA; a S414M or G415H mutation in TynA inducing reporter expression in the presence of Tyra; a G415H mutation in TynA inducing reporter expression in the presence of Tyra; a G415H mutation in TynA inducing reporter expression in the presence of Tyra and PEA; a M83Y mutation in FeaR inducing reporter expression in the presence of PEA; a M83N mutation in FeaR inducing reporter expression in the presence of Tyra; a Fear-KA mutant inducing reporter expression in the presence of Trypta; a tynA-KA or tynA-KP inducing reporter expression in the presence of Trypta; a I109N tynA-KA inducing reporter inducing expression in the presence of Trypta; a I109N FeaR-KA inducing reporter inducing expression in the presence of Trypta; a Q76, Q116, L108(T), W110(S), or W110(C) FeaR mutation; a D413 or Y496 TynA mutation; or PtynA-MG, TynA-KP, or FeaR-KA inducing reporter inducing expression in the presence of Trypta and does not induce expression in the presence of dopamine (DA), phenylethylamine (PEA), or tyramine (Tyra), wherein the FeaR or TynA protein sequence or ligand binding site of FeaR or TynA is at least about 80% identical to a polypeptide encoded by the WT FeaR sequence of SEQ ID NO: 36 or WT TynA sequence of SEQ ID NO: 32 or WT ligand binding site or functional fragment or conservative substitution thereof and has ligand-binding activity.
18. A selective sensor for specifically detecting target aromatic compounds (e.g., Phe, Tyr, PEA, Tyra) comprising: a molecular sensor according to claim 1 or an engineered microorganism (e.g., an E. coli strain) capable of expressing native or non-native FeaR and TynA (or functional fragments, conservative substitutions, mutants, or variants thereof); and a promoter-reporter system comprising a reporter gene under the control of a promoter capable of being induced or repressed by one or more target aromatic compounds (e.g., Phe, Tyr, PEA, Tyra) or enzymatic-reaction product thereof (e.g., aromatic aldehyde, aromatic carboxylic acid) and producing an output response (e.g., increased expression or repression).
19. The selective sensor of claim 18, wherein the output response is a repression of the reporter (e.g., signaling or detection moiety, such as GFP) expression.
20. The selective sensor of claim 18, wherein the output response is an overexpression of the reporter (e.g., signaling or detection moiety, such as GFP).
21. The selective sensor of claim 18, further comprising one or more enzymes or transcription factors.
22. A selective sensor for specifically detecting aromatic amines (e.g., PEA, Tyra, DA, Trypta) or aromatic aldehydes thereof comprising: a TynA and FeaR protein (or a functional mutant or variant thereof); and a reporter gene (comprising a signaling moiety, e.g., GFP) operably linked to an inducible promoter (e.g., PtynA promoter).
23. The selective sensor of claim 22, wherein the selective sensor induces expression of PtynA in response to aromatic aldehydes, but not aromatic amines.
24. The selective sensor of claim 22, wherein the TynA protein or mutant variant thereof is selective to a specific aromatic amine.
25. The selective sensor of claim 22, wherein TynA converts periplasmic amines (e.g., PEA, Tyra, DA, Trypta) to aldehydes which are imported into the cytoplasm; and in the cytoplasm, FeaR induces expression from the PtynA promoter expressing a reporter gene (e.g., detectable signal moiety) when in the presence of aldehydes.
26. The selective sensor of claim 22, wherein the selective sensor is selective to PEA and Tyra.
27. The selective sensor of claim 22, wherein either TynA or FeaR or both are engineered for selectivity.
28. The selective sensor of claim 22, wherein the sensor comprises G494S mutation in TynA and optionally A81T mutation in the FeaR protein (i.e., double mutant sensor (G494S*)).
29. The selective sensor of claim 22, wherein the selective sensor is a Tyra-specific variant, comprises G415H mutation in TynA.
30. The selective sensor of claim 22, wherein G415H mutation in TynA induces expression of a reporter by about 200-fold and/or about 79-fold in response to Tyra and PEA, respectively, with minimal response to DA and Trypta.
31. The selective sensor of claim 22, wherein A81T FeaR mutation with WT TynA is PEA and Tyra non-selective; S414M TynA and WT FeaR is slightly Tyra selective; S141M TynA and A81T FeaR is PEA selective; G415H TynA and WT FeaR is Tyra selective; or G415H TynA and A81T FeaR is PEA selective.
32. The selective sensor of claim 22, wherein FeaR A81L, A81P, A81I, and A81N are PEA-specific variants.
33. The selective sensor of claim 22, wherein Tyra-selective sensors (S414M or G415H TynA) can be transformed into PEA-specific sensors by introducing A81T FeaR; sensor sensitivity (G494S TynA) can be improved by A81T or A81S FeaR mutations; or PEA-Tyra selective sensors (A81T or A81S FeaR) can become PEA-specific sensors when combined with G494S TynA.
34. The engineered molecular sensor of claim 1, wherein the engineered regulator protein is an engineered TrpR and the engineered TrpR comprises one or more of the following mutations: the TrpR variant is O.sub.D or O.sub.1, repressing reporter expression in the presence of Trp and 5-HTP, but not Trypta, wherein the protein sequence or ligand binding site sequence of TrpR is at least about 80% identical to a polypeptide encoded by the WT TrpR sequence of SEQ ID NO: 113 or WT ligand binding site sequence or functional fragment or conservative substitution thereof and has ligand-binding activity.
35. The engineered molecular sensor of claim 1, wherein the reference nucleotide sequence encoding TrpR is SEQ ID NO: 113 and the reference amino acid sequence for TrpR is SEQ ID NO: 122; the reference nucleotide sequence encoding TyrR is SEQ ID NO: 11 and the reference amino acid sequence for TyrR is SEQ ID NO: 124; the reference nucleotide sequence encoding FeaR is SEQ ID NO: 36 and the reference amino acid sequence for FeaR is SEQ ID NO: 125; or the reference nucleotide sequence encoding TynA is SEQ ID NO: 32 and the reference amino acid sequence for TynA is SEQ ID NO: 123.
36. The engineered molecular sensor of claim 1, wherein the engineered regulator protein or enzyme (e.g., TyrR, FeaR, TynA, or TrpR) or the ligand binding site of the engineered regulator protein or enzyme comprises at least 80% identity to a polypeptide encoded by SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 11, or SEQ ID NO: 113; at least 80% identity to SEQ ID NO: 122 (TrpR WT), SEQ ID NO: 123 (tynA WT), SEQ ID NO: 124 (tyrR WT), or SEQ ID NO: 125 (fear WT); or at least 80% identity to D413 to Y496 of SEQ ID NO: 123, C7 to E274 of SEQ ID NO: 124, W12 to S118 of SEQ ID NO: 125, A81 to W110 of SEQ ID NO: 125, Q76 to Q116 of SEQ ID NO: 125, K72 to T83 of SEQ ID NO: 122, or a functional fragment or conservative substitution thereof.
37. The engineered molecular sensor of claim 1, wherein the K.sub.A value of the engineered regulator protein is less than the K.sub.A value of the wild type regulator protein in the presence of a target aromatic compound; the K.sub.A value of the engineered TyrR sensor is between about 0.05 mM and about 0.3 mM optionally in human intestines, serum, or urine; or the K.sub.A value of the engineered TynA-FeaR is between about 0.001 mM and about 0.1 mM optionally in plasma or food.
38. The engineered molecular sensor of claim 1, wherein the presence of a target aromatic compound induces or represses reporter gene expression of the engineered regulator protein or enzyme compared to the wild type regulator protein or enzyme.
39. The engineered molecular sensor of claim 1, wherein the engineered regulator protein or enzyme has a selectivity, induction, or repression response that is greater than wild type.
40. The engineered molecular sensor of claim 1, wherein the regulator protein or enzyme or the regulator protein or enzyme binding site is modified to increase selectivity.
41. The engineered molecular sensor of claim 1, wherein the engineered molecular sensor is a ligand-specific biosensor for phenylalanine, tyrosine, phenylethylamine, or tyramine.
42. The engineered molecular sensor of claim 1, wherein the engineered molecular sensor is directly transferred into probiotic organisms or purified for cell-free sensor application.
43. A selective sensor for specifically detecting target aromatic compounds (e.g., Phe, Tyr) comprising: an engineered microorganism (e.g., an E. coli strain) comprising the engineered molecular sensor according to claim 1 capable of expressing native or non-native TrpR (or functional mutants or variants thereof); and a promoter-reporter system comprising a reporter gene (e.g., GFP) under the control of a promoter (e.g., Ptrp) capable of being induced or repressed by one or more target aromatic compounds and producing an output response (e.g., increased expression or repression of the reporter gene).
44. The selective sensor of claim 43, wherein the TrpR is a TrpR variant O.sub.D or O.sub.1, each with a synthetic Ptrp promoter, the selective sensor having strong repression in the presence of Trp and 5-HTP, but not Trypta.
45. The selective sensor of claim 43, wherein a genomic copy of wild-type trpR is knocked out from the engineered microorganism selected from an E. coli strain (e.g., EcN).
46. The selective sensor of claim 43, wherein the WT TrpR system has strong repression of GFP expression with fold repressions of 120-fold, 20-fold, and 7-fold in response to Trp, 5-HTP, and Trpta, respectively.
47. The selective sensor of claim 43, wherein the engineered TrpR variants maintain strong repression in the presence of Trp and 5-HTP, but not Trypta.
48. The selective sensor of claim 44, wherein the O.sub.D variant demonstrates about 5-fold and about 7-fold repression in response to Trp and 5-HTP, respectively.
49. The selective sensor of claim 44, wherein the O.sub.1 variant demonstrates about 60-fold and about 15-fold repression in response to Trp and 5-HTP, respectively.
50. The selective sensor of claim 43, wherein the target aromatic compounds are one or more of tryptophan (Trp), 5-hydroxytryptophan (5-HTP), or tryptamine (Trypta).
51. An artificial DNA construct comprising, as operably associated components in the 5′ to 3′ direction of transcription: (a) a promoter functional in a microorganism (e.g., transgenic microorganism, wild type microorganism); (b) a first polynucleotide comprising a nucleotide sequence encoding (i) a first polypeptide having TrpR activity; (ii) a second polypeptide having TyrR activity; or (iii) a first polypeptide having FeaR activity and a second polynucleotide comprising a nucleotide sequence encoding a second polypeptide having TynA activity; (c) a reporter gene (e.g., GFP); and (d) a transcriptional termination sequence; wherein, the microorganism is capable of expressing native or non-native (i) TrpR; (ii) TyrR; or (iii) FeaR and TynA (or functional mutants or variants thereof); and the microorganism specifically expresses or represses reporter gene expression compared to a microorganism not comprising the artificial DNA construct in the presence or absence of aromatic compounds.
52. A microbial sensor selected from an engineered wild type or transgenic microorganism transformed with the artificial DNA construct of claim 51.
53. The microbial sensor of claim 52, wherein the wild type or transgenic microorganism is selected from Escherichia coli Nissle 1917 (EcN), DH10B, or E. coli MG1655.
54. The microbial sensor of claim 52, wherein the selective sensor is selective for an aromatic compound and the aromatic compound is an aromatic amino acid selected from phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), and combinations thereof.
55. The microbial sensor of claim 52, wherein the selective sensor is selective for an aromatic compound and the aromatic compound is an aromatic amine neurochemical selected from dopamine (DA), phenylethylamine (PEA), tyramine (Tyra), tryptamine (Trypta), serotonin, epinephrine, or norepinephrine.
56. The microbial sensor of claim 52, wherein a TrpR-based sensor is selective for Trp; a TyrR-based sensor is selective for Phe and Tyr; or a TynA-FeaR sensor system is selective for aromatic amines.
57. A ligand-specific sense-and-respond system, comprising purified sensors or engineered proteins or probiotics for specific sensing of aromatic compounds (e.g., amino acids, aromatic amines, aromatic neurochemicals) comprising: providing a orthogonal DNA-TF binding system with accompanying selectivity changes; changing ligand-TF binding specificity by leveraging differential multimerization patterns of TyrR without affecting DNA-TF binding interaction; or a “dual-control knob” strategy to improve the specificity and sensitivity of substrate-enzyme and ligand-TF interaction while maintaining DNA-TF binding interaction.
58. The ligand-specific sense-and-respond system of claim 57, wherein target ligands are structurally similar and ligand-protein binding controls downstream functions such as reporter gene expression.
59. The ligand-specific sense-and-respond system of claim 57, comprising an engineered microorganism.
60. A method of using the engineered molecular sensor of claim 1, comprising obtaining or having obtained a biological sample from a subject and contacting the biological sample with the engineered molecular sensor.
61. The method of claim 60, wherein the subject has an aromatic compound-associated disease, disorder, or condition.
62. The method of claim 60, wherein elevated levels of Phe detected by the sensor indicate the subject has phenylketonuria.
63. The method of claim 60, wherein elevated levels of Tyr detected by the sensor indicate the subject has type 2 tyrosinemia.
64. The method of claim 60, wherein elevated levels of PEA detected by the sensor indicate the subject has a psychological disorder.
65. The method of claim 60, wherein the presence of Tyra detected by the sensor indicates catecholamine release and an increase in blood pressure.
66. The method of claim 60, wherein the presence of Trypta detected by the sensor causes serotonin release and stimulation of gastrointestinal motility.
67. A method of using the engineered molecular sensor of claim 1, comprising monitoring food quality or diagnosing or treating metabolic, digestive, or neurological disorders.
68. The method of claim 67, wherein the presence of PEA, Tyra, or Trypta in food detected by the sensor indicates microbial contamination.
69. The engineered molecular sensor of claim 1, wherein the sensor dynamically identifies microbial contamination in consumable products, manages various debilitating neurological disorders, or normalizes dysregulated metabolites associated with metabolic disorders.
70. The engineered molecular sensor of claim 1, wherein the sensor recognizes or is selective for aromatic metabolites associated with various metabolic or neurological disorders or medical conditions.
71. The engineered molecular sensor of claim 70, wherein the aromatic compounds are selected from phenylalanine (Phe) or tyrosine (Tyr).
72. The engineered molecular sensor of claim 70, wherein the aromatic compounds are neurochemicals.
73. The engineered molecular sensor of claim 72, wherein the neurochemicals are selected from aromatic neurotransmitters or neuromodulators.
74. The engineered molecular sensor of claim 72, wherein the neurochemicals are selected from dopamine (DA), phenylethylamine (PEA), tyramine (Tyra), tryptamine (Trypta), serotonin, epinephrine, or norepinephrine.
75. The method of claim 60, wherein the subject has or is suspected of having a medical condition associated with elevation, presence, or absence of aromatic compounds.
76. The engineered molecular sensor of claim 1, wherein the sensor differentiates metabolites with divergent functions even having structural similarity.
77. The engineered molecular sensor of claim 1, wherein the sensor modulates the specificity of ligand-protein binding while maintaining protein-DNA interactions and downstream gene expression control.
78. A method of using the engineered molecular sensor of claim 1, the method comprising monitoring food quality, diagnosing or treating metabolic, digestive, or neurological disorders in probiotics or ex vivo wearable, paper-based or cell-free systems, or dynamically regulating enzymatic pathways for microbial metabolic engineering using the engineered molecular sensor.
79. A method of protein engineering (e.g., a regulator protein or enzyme) comprising: mutagenizing specific amino acids in and around a ligand-binding site of a protein or enzyme (e.g., TrpR, TyrR, FeaR, TynA), wherein the mutagenizing enables changes in ligand-protein binding specificity while maintaining protein-DNA interaction and thus downstream gene expression control; and linking ligand-protein binding to output response (e.g., promoter-reporter gene system).
Description
DESCRIPTION OF THE DRAWINGS
[0007] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
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DETAILED DESCRIPTION
[0041] The present disclosure is based, at least in part, on the development of the first ligand-specific or selective sensors for aromatic compounds such as aromatic neurochemicals or aromatic amino acids.
[0042] Examples of aromatic amino acids can be phenylalanine (Phe) and tyrosine (Tyr). Examples of aromatic neurochemicals (e.g., neurotransmitters, neuromodulators) can be aromatic amines, such as phenylethylamine (PEA) and tyramine (Tyra), which are structurally similar and have been implicated in a variety of medical conditions.
[0043] As described herein, biosensors with high selectivity for aromatic compounds, such as phenylalanine, tyrosine, phenylethylamine, or tyramine, were developed and demonstrated for the first time.
[0044] Applications for this technology can include:
[0045] 1) developing medically-relevant probiotic sensors with high specificity, which is critical to differentiate metabolites with divergent functions and create smart probiotics for accurate diagnostic tools; and
[0046] 2) developing autonomous microbes that dynamically identify microbial contamination in consumable products, manage various debilitating neurological disorders, and normalize dysregulated metabolites associated with metabolic disorders.
[0047] Publically available or computational tools purchased include: the MOTIF Search webtool (genome.jp), Robetta Web Server, Chem3D 16.0 (PerkinElmer, Waltham, Mass.), Rosetta Ligand Docking Server hosted by ROSIE, KEGG database, https://www.hmpdacc.org/HMRGD/, Interactive tree of life (iTOL) phylogeny tree annotation tool, HMP database, Greengenes, NCBI, and PyMOL 2.4.1 (Schrödinger, Inc., New York, N.Y.).
[0048] Engineered Microorganism
[0049] Many probiotics have been engineered for therapeutic applications, including treating various cancers, inflammatory disorders, metabolic disorders, and bacterial infections. Many of these probiotics constitutively express the proteins, raising concerns of off-target effects and genetic stability. Several engineered probiotics partially mitigate this concern by using sensors that limit protein expression to targeted biogeographical regions. For example, probiotics have been designed to regulate the therapeutic expression through sensors that detect oxygen content. However, they should also be engineered with biosensors that recognize the disease state by measuring the concentrations of relevant metabolites. By using disease-specific sensors, therapeutics can be delivered with both geographical and temporal precision. In addition, these sensors expand the potential applications of the engineered probiotics, which can be used to diagnose numerous disorders.
[0050] To develop these smart probiotics, the toolbox of sensors needs to be expanded for common probiotic microbes. Synthetic sensors can be created using several protein design approaches. However, sensors can also be obtained more efficiently by mining sensors that naturally exist in microbes. These sensors can often be directly transferred into probiotic organisms for use in various applications. However, most natural sensors recognize multiple structurally similar, but functionally diverse, metabolites often found in close proximity. To accurately correlate the concentration of a chemical with biological outcomes, engineered microbes need to differentiate between different chemicals. In addition, sensors often have an inadequate sensitivity to the ligand or lack orthogonality with native regulatory pathways in the probiotic, limiting their utility.
[0051] Specific sensing is critical for many probiotic sensor applications. Chronically elevated levels of structurally similar phenylalanine (Phe) and tyrosine (Tyr) are associated with the distinct disorders phenylketonuria and type 2 tyrosinemia, respectively. Sensors for both metabolites have been generated in different E. coli strains. However, the sensors are based on the wild-type version of the multi-ligand responsive TyrR transcription factor from E. coli and have limited selectivity and low dynamic ranges, making them unsuitable for differentiating between the two disorders. Similarly, the structurally similar amines phenylethylamine (PEA), tyramine (Tyra), and tryptamine (Trypta) are all commonly found and produced in the gut, but contribute to distinct biological outcomes. For example, extreme levels of PEA have been associated with a variety of psychological disorders, the presence of Tyra leads to catecholamine release and an increase in blood pressure, and the presence of Trypta causes serotonin release and the stimulation of gastrointestinal motility. Additionally, the presence of PEA, Tyra, and Trypta in food are indicators of microbial contamination, and eating foods with high levels of Tyra can lead to poisoning. Currently, there are no biosensors with high ligand specificity for these medically relevant chemicals, which can be employed in probiotic microbes.
[0052] A variety of protein engineering methods have been demonstrated for optimizing the selectivity and sensitivity of protein and RNA sensors. These approaches include directed evolution, rational design, and computational de novo design. Although each method has advantages, rational engineering can uniquely be performed using both basic knowledge of the protein structure and small library sizes, allowing for rapid construction and screening of libraries. In addition, when the structure of the protein is unknown, conserved or essential residues can also be identified through computational simulations or by aligning the sequence of the sensor with other proteins in the same family. Despite advances in protein engineering, creating ligand-specific sense-and-respond systems remains challenging due to the need to couple subtle protein conformational changes with differential protein-DNA interactions and gene expression control, especially when the target ligands are structurally similar.
[0053] Here is described the generation of sensors for a variety of disease-relevant aromatic metabolites in probiotic Escherichia coli Nissle 1917 (EcN). These metabolites include aromatic amino acids Phe, Tyr, and tryptophan (Trp), which are required to synthesize proteins and diverse essential metabolites, and aromatic amine neurochemicals dopamine (DA), PEA, Tyra, and Trypta, which are associated with many health issues. To generate sensors for these aromatic metabolites, three sensor systems were identified and engineered: the TrpR (Trp) sensor, the TyrR (Phe and Tyr) sensor, and the TynA-FeaR (aromatic amine) sensor system. Multiple engineered TrpR sensors were first characterized, which were previously created to be orthogonal to the wild-type E. coli-native system, and how the mutations impact the selectivity of the sensors was assessed. Next, the ligand selectivity of the TyrR and TynA-FeaR sensor systems was engineered and their sensitivity was tuned by rationally selecting and individually mutating amino acids in TyrR, TynA, and FeaR. This method of protein engineering quickly generates multiple small libraries that can be efficiently screened. Altogether, the first sensors selective for Phe, Tyr, PEA, or Tyra were generated. In engineering FeaR, novel insights into the uncharacterized structure of FeaR are also provided and residues important for ligand binding were identified for the first time. This work provides sensors with diverse clinical applications in engineering smart probiotics as well as a generalizable approach to modulating the specificity of ligand-protein binding while maintaining protein-DNA interactions and thus downstream gene expression control.
[0054] Microorganism
[0055] The disclosed system uses a microorganism (e.g., probiotic (bacteria, yeast)) to display biomolecules or binding agents.
[0056] Generally, one of the criteria that bacteria must meet in order for them to be regarded as probiotics is that they have to be able to survive and thrive throughout the GIT conditions and confer their beneficial effects. A preferred microorganism does not colonize the gut, and is thus, easily controlled.
[0057] For example, the microorganism can be Escherichia coli Nissle 1917 (EcN), DH10B, or E. coli MG1655. Other microorganisms that can engineered to incorporate sensors can be probiotics known in the art (see e.g., Bober Synthetic Biology Approaches to Engineer Probiotics and Members of the Human Microbiota for Biomedical Applications. Annu Rev Biomed Eng. 2018; 20:277-300; Mathipa and Thantsha Gut Pathog (2017) 9:28). Probiotics can include bacteria from the genera Streptococcus, Enterococcus, Pediococci, Weissella, or Lactococcus, but the most common ones used belong to Lactobacillus and Bifidobacteria spp.
[0058] The microorganism can be yeast, such as a Saccharomyces (e.g., Saccharomyces cerevisiae, Saccharomyces boulardii CNCM 1-745). Due to its inability to colonize the human gut, S. boulardii can be engineered to act as a sensor (and can pass through a host gut).
[0059] Binding Agents/Biomolecules
[0060] Binding agents, such as biomolecules (e.g., a ligand-binding moiety) can be displayed on the surface of the engineered microorganism. As such, the engineered microorganism can be designed to have a binding affinity to a specific aromatic compound with discriminating specificity.
[0061] The binding moiety can be the proteins, TrpR (Trp) sensor, the TyrR (Phe and Tyr) sensor, and the TynA-FeaR (aromatic amine) sensor system. Here, it is shown that their sensitivity can be tuned by rationally selecting and individually mutating amino acids in TyrR, TynA, and FeaR. These sensors have been shown to be selective for Phe, Tyr, PEA, or Tyra.
[0062] K.sub.A is a half maximal constant. K.sub.A values can be greater than 0 mM, between about 0.001 mM and 0.3 mM. For example, for a TyrR in human intestines, serum, or urine a K.sub.A of 0.05-0.3 mM is considered sufficient. As another example, in plasma or food, a K.sub.A of 0.001-0.1 mM is considered sufficient.
[0063] TrpR (Trp) Sensor.
[0064] First, multiple engineered TrpR sensors were characterized, which were previously created to be orthogonal to the wild-type E. coli-native system, and assess how the mutations impact the selectivity of the sensors.
[0065] Transcriptional Regulatory Protein TyrR (Phe and Tyr) Sensor.
[0066] The ligand selectivity of the TyrR sensor was engineered and their sensitivity was tuned by rationally selecting and individually mutating amino acids in TyrR.
[0067] As shown in Example 1, the Phe-specific sensors have the potential to be applied to kinetically diagnose and treat disorders that cause Phe-dysregulation without interference from intestinal Tyr.
[0068] TyrR protein is involved in transcriptional regulation of aromatic amino acid biosynthesis and transport. TyrR modulates the expression of at least 8 unlinked operons. Seven of these operons are regulated in response to changes in the concentration of the three aromatic amino acids (phenylalanine, tyrosine and tryptophan). These amino acids are suggested to act as co-effectors which bind to the TyrR protein to form an active regulatory protein. In most cases TyrR causes negative regulation, but positive effects on the tyrP gene have been observed at high phenylalanine concentrations. The native tyrR gene (E. coli) is autogenously regulated by a mechanism that gives similar rates of expression of tyrR irrespective of the concentration of the aromatic amino acids.
[0069] Monoamine Oxidase TynA-FeaR Regulator (Aromatic Amine) Sensor System.
[0070] The ligand selectivity of the TynA-FeaR sensor systems was engineered and sensitivity was tuned by rationally selecting and individually mutating amino acids in TynA and FeaR.
[0071] TynA-FeaR system: a sensor plasmid, which consisted of constitutively expressed TynA and FeaR from E. coli MG1655, and a reporter plasmid, which consisted of GFP under the control of PtynA promoter (see e.g.,
[0072] Target Aromatic Compounds
[0073] As described herein, the present disclosure provides for a biosensor that specifically interacts with aromatic compounds.
[0074] For example, the aromatic compound can be an aromatic amino acid selected from phenylalanine (Phe), tyrosine (Tyr), or tryptophan (Trp).
[0075] As another example, the aromatic compound can be an aromatic amine neurochemical selected from dopamine (DA), phenylethylamine (PEA), tyramine (Tyra), tryptamine (Trypta), serotonin, epinephrine, or norepinephrine.
[0076] Reporters for Artificial Sensors/Signaling System
[0077] The engineered microorganism can be engineered to incorporate reporters for use in artificial sensing and signaling.
[0078] The engineered microorganisms described herein can comprise a reporter (or sensor). The reporter can be a sensor protein to detect pH, an immune receptor, or reporter proteins (e.g., GFP, RFP (e.g., mCherry), BFP, luminescence protein (e.g., luciferase)).
[0079] For example, the engineered microorganism can comprise a promoter-reporter system and/or a dual-control knob system.
[0080] Biosensors/Detectors
[0081] The present disclosure also provides for a method of constructing engineered microorganism biosensors that selectively recognize and react to a wide range of sensible target aromatic compounds in vivo. The engineered microorganism can include a reporter. The three sensor systems described here include the TrpR (Trp) sensor, the TyrR (Phe and Tyr) sensor, and the TynA-FeaR (aromatic amine) sensor system.
[0082] As described herein, the engineered microorganisms can be used for sensing applications. For example, a portion of a functional protein can be used to induce or repress a transcriptional response.
[0083] The promoter PtyrP upregulates gene expression via the phenylalanine-binding transcriptional dual regulator TyrR.
[0084] As described in Example 1, six E. coli-native promoters were characterized using genomic expression or plasmid-based overexpression of TyrR to identify the best promoter in EcN. PtyrP was selected as a sensor as it displayed an activating response to Phe and a repressing response to Tyr, allowing for clear differentiation between the presence of each amino acid, especially with overexpression of TyrR (see e.g.,
[0085] As described herein, engineered microorganisms that can be used for detecting or sensing aromatic compounds. For example, a detection moiety can be used (e.g., GFP, RFP, BFP, luminescence protein). As such, every engineered microorganism that sensed a target aromatic compound will overexpress (“light up”) or be repressed.
[0086] As a microbial biosensor targeting gut health, the disclosed engineered E. coli can overcome limitations of conventional approaches that depend on very limited existing sensing mechanisms in nature.
[0087] Molecular Engineering
[0088] The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
[0089] The term “transfection,” as used herein, refers to the process of introducing nucleic acids into cells by non-viral methods. The term “transduction,” as used herein, refers to the process whereby foreign DNA is introduced into another cell via a viral vector.
[0090] The terms “heterologous DNA sequence”, “exogenous DNA segment”, or “heterologous nucleic acid,” as used herein, each refers to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling or cloning. The terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides. A “homologous” DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.
[0091] Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.
[0092] An “expression vector”, otherwise known as an “expression construct”, is generally a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins. The vector is engineered to contain regulatory sequences that act as enhancer and/or promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the efficient production of protein, and this may be achieved by the production of significant amount of stable messenger RNA, which can then be translated into protein. The expression of a protein may be tightly controlled, and the protein is only produced in significant quantity when necessary through the use of an inducer, in some systems however the protein may be expressed constitutively. As described herein, Escherichia coli is used as the host for protein production, but other cell types may also be used.
[0093] In molecular biology, an “inducer” is a molecule that regulates gene expression. An inducer can function in two ways, such as:
[0094] (i) By disabling repressors. The gene is expressed because an inducer binds to the repressor. The binding of the inducer to the repressor prevents the repressor from binding to the operator. RNA polymerase can then begin to transcribe operon genes.
[0095] (ii) By binding to activators. Activators generally bind poorly to activator DNA sequences unless an inducer is present. An activator binds to an inducer and the complex binds to the activation sequence and activates the target gene. Removing the inducer stops transcription. Because a small inducer molecule is required, the increased expression of the target gene is called induction.
[0096] Repressor proteins bind to the DNA strand and prevent RNA polymerase from being able to attach to the DNA and synthesize mRNA. Inducers bind to repressors, causing them to change shape and preventing them from binding to DNA. Therefore, they allow transcription, and thus gene expression, to take place.
[0097] For a gene to be expressed, its DNA sequence must be copied (in a process known as transcription) to make a smaller, mobile molecule called messenger RNA (mRNA), which carries the instructions for making a protein to the site where the protein is manufactured (in a process known as translation). Many different types of proteins can affect the level of gene expression by promoting or preventing transcription. In prokaryotes (such as bacteria), these proteins often act on a portion of DNA known as the operator at the beginning of the gene. The promoter is where RNA polymerase, the enzyme that copies the genetic sequence and synthesizes the mRNA, attaches to the DNA strand.
[0098] Some genes are modulated by activators, which have the opposite effect on gene expression as repressors. Inducers can also bind to activator proteins, allowing them to bind to the operator DNA where they promote RNA transcription. Ligands that bind to deactivate activator proteins are not, in the technical sense, classified as inducers, since they have the effect of preventing transcription.
[0099] A “promoter” is generally understood as a nucleic acid control sequence that directs transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates transcription of an operably linked gene in response to a particular stimulus. A promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
[0100] A “ribosome binding site”, or “ribosomal binding site (RBS)”, refers to a sequence of nucleotides upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome during the initiation of translation. Generally, RBS refers to bacterial sequences, although internal ribosome entry sites (IRES) have been described in mRNAs of eukaryotic cells or viruses that infect eukaryotes. Ribosome recruitment in eukaryotes is generally mediated by the 5′ cap present on eukaryotic mRNAs.
[0101] A “transcribable nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of being transcribed into an RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).
[0102] The “transcription start site” or “initiation site” is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein encoding sequences in the 3′ direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5′ direction) are denominated negative.
[0103] “Operably-linked” or “functionally linked” refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation. The two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates transcription of the gene of interest in a cell.
[0104] A “construct” is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.
[0105] A construct of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3′ transcription termination nucleic acid molecule. In addition, constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3′-untranslated region (3′ UTR). Constructs can include but are not limited to the 5′ untranslated regions (5′ UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.
[0106] The term “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragments are referred to as “transgenic” cells, and organisms comprising transgenic cells are referred to as “transgenic organisms”.
[0107] “Transformed,” “transgenic,” and “recombinant” refer to a host cell or organism such as a bacterium, cyanobacterium, animal, or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like. The term “untransformed” refers to normal cells that have not been through the transformation process.
[0108] “Wild-type” refers to a virus or organism found in nature without any known mutation. A “wild type” organism can be genetically engineered to modulate native or non-native gene expression resulting in a transgenic or engineered organism.
[0109] Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above-required percent identities and retaining a required activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991) Gene 97(1), 119-123; Ghadessy et al. (2001) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide and/or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.
[0110] Nucleotide and/or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2, or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity=XN100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A. For example, the percent identity to a reference sequence (e.g., binding pocket, entire protein, or functional fragment thereof) can be at least 80% or about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.
[0111] Substitution refers to the replacement of one amino acid with another amino acid in a protein or the replacement of one nucleotide with another in DNA or RNA. Insertion refers to the insertion of one or more amino acids in a protein or the insertion of one or more nucleotides with another in DNA or RNA. Deletion refers to the deletion of one or more amino acids in a protein or the deletion of one or more nucleotides with another in DNA or RNA. Generally, substitutions, insertions, or deletions can be made at any position so long as the required activity is retained.
[0112] So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example, the exchange of Glu by Asp, Gln by Asn, Val by Ile, Leu by Ile, and Ser by Thr. For example, amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine), hydroxyl or sulfur/selenium-containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine). Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. An amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of these artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.
[0113] “Highly stringent hybridization conditions” are defined as hybridization at 65° C. in a 6×SSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (T.sub.m) of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65° C. in the salt conditions of a 6×SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65° C. in the same salt conditions, then the sequences will hybridize. In general, the melting temperature for any hybridized DNA:DNA sequence can be determined using the following formula: T.sub.m=81.5° C.+16.6(log.sub.10[Na.sup.+])+0.41(fraction G/C content)−0.63(% formamide)−(600/1). Furthermore, the T.sub.m of a DNA:DNA hybrid is decreased by 1-1.5° C. for every 1% decrease in nucleotide identity (see e.g., Sambrook and Russel, 2006).
[0114] Host cells can be transformed using a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transformed cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.
TABLE-US-00001 Conservative Substitutions I Side Chain Characteristic Amino Acid Aliphatic Non-polar G A P I L V Polar-uncharged C S T M N Q Polar-charged D E K R Aromatic H F W Y Other N Q D E
TABLE-US-00002 Conservative Substitutions II Side Chain Characteristic Amino Acid Non-polar (hydrophobic) A. Aliphatic: A L I V P B. Aromatic: F W C. Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl: S T Y B. Amides: N Q C. Sulfhydryl: C D. Borderline: G Positively Charged (Basic): K R H Negatively Charged (Acidic): D E
TABLE-US-00003 Conservative Substitutions III Original Residue Exemplary Substitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln, His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H) Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val, Met, Ala, Phe Lys (K) Arg, Gln, Asn Met(M) Leu, Phe, Ile Phe (F) Leu, Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp(W) Tyr, Phe Tyr (Y) Trp, Phe, Tur, Ser Val (V) Ile, Leu, Met, Phe, Ala
[0115] Exemplary nucleic acids that may be introduced to a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods. The term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA that is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.
[0116] Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).
[0117] Methods of down-regulation or silencing genes are known in the art. For example, expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides (ASOs), protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), and micro RNAs (miRNA) (see e.g., Rinaldi and Wood (2017) Nature Reviews Neurology 14, describing ASO therapies; Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326-330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401-423, describing RNAi). RNAi molecules are commercially available from a variety of sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinformatics & Research Computing). Traits influential in defining optimal siRNA sequences include G/C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3′ overhangs.
[0118] Genome Editing
[0119] As described herein, expression of various signals (e.g., mutants, proteins) can be modulated (e.g., reduced, eliminated, or enhanced) using genome editing. Processes for genome editing are well known; see e.g. Aldi 2018 Nature Communications 9 (1911). Except as otherwise noted herein, therefore, the process of the present disclosure can be carried out in accordance with such processes.
[0120] For example, genome editing can comprise CRISPR/Cas9, CRISPR-Cpf1, TALEN, or ZNFs. Adequate blockage of various signals by genome editing can result in engineered sensors for detection of aromatic compounds, for example.
[0121] As an example, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are a new class of genome-editing tools that target desired genomic sites in mammalian cells. Recently published type II CRISPR/Cas systems use Cas9 nuclease that is targeted to a genomic site by complexing with a synthetic guide RNA that hybridizes to a 20-nucleotide DNA sequence and immediately preceding an NGG motif recognized by Cas9 (thus, a (N).sub.20NGG target DNA sequence). This results in a double-strand break three nucleotides upstream of the NGG motif. The double strand break instigates either non-homologous end-joining, which is error-prone and conducive to frameshift mutations that knock out gene alleles, or homology-directed repair, which can be exploited with the use of an exogenously introduced double-strand or single-strand DNA repair template to knock in or correct a mutation in the genome. Thus, genomic editing, for example, using CRISPR/Cas systems could be useful tools for targeting cells by the removal or addition of various signals (e.g., to activate (e.g., CRISPRa), upregulate, downregulate).
[0122] For example, the methods as described herein can comprise a method for altering a target polynucleotide sequence in a cell comprising contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein.
[0123] Examples of engineering a probiotic, for example, can include inserting a functional gene with a viral vector.
[0124] Any vector known in the art can be used. For example, the vector can be a viral vector selected from retrovirus, lentivirus, herpes, adenovirus, adeno-associated virus (AAV), rabies, Ebola, lentivirus, or hybrids thereof.
Gene Editing Strategies
[0125]
TABLE-US-00004 Viral Vectors Strategy Retroviruses Retroviruses are RNA viruses transcribing their single- stranded genome into a double-stranded DNA copy, which can integrate into host chromosome Adenoviruses (Ad) Ad can transfect a variety of quiescent and proliferating cell types from various species and can mediate robust gene expression Adeno-associated Recombinant AAV vectors contain no viral DNA and can Viruses (AAV) carry ~4.7 kb of foreign transgenic material. They are replication defective and can replicate only while coinfecting with a helper virus plasmid DNA pDNA has many desired characteristics as a gene (pDNA) therapy vector; there are no limits on the size or genetic constitution of DNA, it is relatively inexpensive to supply, and unlike viruses, antibodies are not generated against DNA in normal individuals RNAi RNAi is a powerful tool for gene specific silencing that could be useful as an enzyme reduction therapy or means to promote read-through of a premature stop codon
[0126] Aromatic Compound-Associated Diseases, Disorders, or Conditions
[0127] The sensors, systems, and methods described herein can be used for the detection or monitoring of aromatic compound-associated diseases, disorders, or conditions.
[0128] For example, elevated levels of structurally similar Phe and Tyr are associated with the distinct disorders phenylketonuria and type 2 tyrosinemia, respectively. As another example, extreme levels of PEA have been associated with a variety of psychological disorders, the presence of Tyra leads to catecholamine release and an increase in blood pressure, and the presence of Trypta causes serotonin release and the stimulation of gastrointestinal motility. Additionally, the presence of PEA, Tyra, and Trypta in food are indicators of microbial contamination, and eating foods with high levels of Tyra can lead to poisoning. Currently, there are no biosensors with high ligand specificity for these chemicals.
[0129] Serotonin is a chemical that the body produces naturally. It's needed for the nerve cells and brain to function. But too much serotonin can cause signs and symptoms that can range from mild (shivering or diarrhea) to severe (muscle rigidity, fever, or seizures). Severe serotonin syndrome can cause death if not treated. Serotonin syndrome can be a result of too much serotonin in your body. It is usually caused by taking drugs or medications that affect serotonin levels. Stopping the drug(s) or medication(s) causing serotonin syndrome is the main treatment.
[0130] Many of these metabolites, including DA, PEA, Tyra, and Trypta can be found in the intestines. As such, selective detection of such compounds is needed.
[0131] Formulation
[0132] The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington's Pharmaceutical Sciences (A. R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
[0133] The term “formulation” refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a “formulation” can include pharmaceutically acceptable excipients, including diluents or carriers.
[0134] The term “pharmaceutically acceptable” as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Md., 2005 (“USP/NF”), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP/NF, etc. may also be used.
[0135] The term “pharmaceutically acceptable excipient,” as used herein, can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
[0136] A “stable” formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0° C. and about 60° C., for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.
[0137] The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic, or other physical forces.
[0138] Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled-release preparations can also be used to affect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently, affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled-release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.
[0139] Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition.
[0140] Administration
[0141] Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.
[0142] As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implanted, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intracerebroventricular, subcutaneous, intranasal, epidural, intrathecal, ophthalmic, transdermal, buccal, and rectal.
[0143] Agents and compositions described herein can be administered in a variety of methods well known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 μm), nanospheres (e.g., less than 1 μm), microspheres (e.g., 1-100 μm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.
[0144] Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.
[0145] Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule/agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo; prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency; improve taste of the product; or improve shelf life of the product.
[0146] Kits
[0147] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to engineered probiotics, engineered protein regulators, and components for making and using same. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.
[0148] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal, or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.
[0149] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or another substrate, and/or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet website specified by the manufacturer or distributor of the kit.
[0150] A control sample or a reference sample as described herein can be a sample from a healthy subject or sample, a wild-type subject or sample, or from populations thereof. A reference value can be used in place of a control or reference sample, which was previously obtained from a healthy subject, a group of healthy subjects, or a wild-type subject or sample. A control sample or a reference sample can also be a sample with a known amount of a detectable compound or a spiked sample.
[0151] Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).
[0152] Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
[0153] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term “about.” In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. The recitation of discrete values is understood to include ranges between each value.
[0154] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term “or” as used herein, including the claims, is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.
[0155] The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that “comprises,” “has” or “includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.
[0156] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.
[0157] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
[0158] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.
[0159] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.
EXAMPLES
[0160] The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.
Example 1: Engineering Ligand-Specific Biosensors for Aromatic Amino Acids and Neurochemicals
[0161] This work represents a considerable achievement and includes the first ligand-specific or ligand-selective sensors for aromatic amino acids such as phenylalanine (Phe) and tyrosine (Tyr) as well as neurochemicals such as phenylethylamine (PEA) and tyramine (Tyra), which are structurally similar and have been implicated in a variety of medical conditions. Importantly, it lays the groundwork for developing medically relevant probiotic sensors with high specificity, which is critical to differentiate metabolites with divergent functions and create smart probiotics for accurate diagnostic tools.
[0162] Microbes have evolved diverse sensor systems that detect metabolites of great medical importance. These sensors have the potential to be utilized in probiotic microbes to provide diagnostic information and deliver therapeutics with temporal and geographical precision. However, microbial sensors found in nature often have promiscuity to several structurally similar aromatic amino acids, common neurotransmitters, or other neuromodulators, limiting their practical applications. Although similar, these metabolites have significantly different functions. For example, chronically elevated levels of structurally similar Phe and Tyr are associated with the distinct disorders phenylketonuria and type 2 tyrosinemia, respectively. Extreme PEA levels have been associated with a variety of psychological disorders, while the presence of Tyra leads to catecholamine release and an increase in blood pressure. Due to such differences in associated diseases and functions, specific sensing is critical for probiotic sensor applications. Despite advances in protein engineering for specific ligand-protein interactions, engineering ligand-specific sense-and-respond systems remain challenging, especially when the target ligands are structurally similar and when ligand-protein binding should control downstream functions such as gene expression. This is mainly due to the challenge in coupling subtle protein conformational changes caused by binding of similar ligands with differential DNA interactions.
[0163] In this work, two promiscuous sensors are characterized that recognize aromatic metabolites associated with various metabolic and neurological disorders. Common methods of protein engineering require extensive structural knowledge of the proteins or massive library sizes. In contrast, to improve ligand selectivity, the responsible proteins, including TyrR, TynA, and FeaR, were rationally engineered by identifying and individually mutagenizing specific amino acids in and around the ligand-binding sites of these sensors. From these three case studies, this simple and generalizable method of protein engineering is shown to be effective and time-efficient, require small library sizes with only a basic understanding of the protein structure, and enables changes in ligand-protein binding specificity while maintaining protein-DNA interaction and thus downstream gene expression control.
[0164] Protein engineering for ligand-protein interaction has been extensively performed. However, linking ligand-protein binding to output response remains challenging. Current approaches include designing proteins that fluoresce upon ligand binding or employing ligand-binding transcription factors (TFs). While the former can be used to create sensors, the latter can generate sensors or signal-responsive controllers for gene expression. Because this TF-based system requires the maintenance or engineering of DNA-protein interaction in addition to engineering ligand-protein binding, engineering the TF-based system has been challenging, especially when the target ligands are structurally similar. To address this issue, demonstrated herein is an approach to change ligand-TF binding specificity by leveraging differential multimerization patterns of TyrR without affecting DNA-TF binding interaction (see e.g.,
[0165] The ligand binding site for TynA comprises at least a functional portion of SEQ ID NO: 32, having catalytic residues D413 and Y496. The ligand binding site for FeaR comprises at least a functional portion of SEQ ID NO: 36, wherein the ligand binding region is from W12 to S118 cover by the magenta region in
[0166] In addition, the computational and experimental analyses of FeaR provide novel insights into the otherwise uncharacterized structure of FeaR. The location of the FeaR ligand-binding pocket and potentially critical residues in ligand binding are identified herein for the first time. This approach allows for the generation of a highly efficient, specific, and sensitive sensor for PEA. In addition, residues that can be mutagenized in future work to generate novel ligand-selective sensors for several additional aromatic neurotransmitters and neuromodulators are also identified herein.
[0167] The novel ligand-selective sensors generated in this work will have diverse applications in synthetic biology. They can be applied to engineer autonomous microbes that dynamically identify microbial contamination in consumable products, manage various debilitating neurological disorders, and normalize dysregulated metabolites associated with metabolic disorders. In addition, the protein engineering methods demonstrated here can be widely applied to develop enzymes and sensors with applications in bioenergy, commodity chemicals, bioremediation, and healthcare.
[0168] Summary
[0169] Microbial biosensors have diverse applications in metabolic engineering and medicine. Specific and accurate quantification of chemical concentrations allows for adaptive regulation of enzymatic pathways and temporally precise expression of diagnostic reporters. Although biosensors should differentiate structurally similar ligands with distinct biological functions, such specific sensors are rarely found in nature and are challenging to create. Using E. coli Nissle 1917, generally regarded as a safe microbe, two biosensor systems that promiscuously recognize aromatic amino acids or neurochemicals were characterized. To improve the sensors' selectivity and sensitivity, rational protein engineering was applied by identifying and mutagenizing amino acid residues and the ligand-specific biosensors for phenylalanine, tyrosine, phenylethylamine, and tyramine were successfully demonstrated (see e.g.,
[0170] Introduction
[0171] Microbial biosensors can be utilized to kinetically regulate and quantify the products of metabolic pathways, diagnose diseases in vivo in probiotics, and analyze ex vivo samples through wearable, paper-based, and cell-free systems. Synthetic biological sensors can be created using several protein design approaches. However, sensors can also be obtained more efficiently by mining sensors that naturally exist in microbes. These sensors can often be directly transferred into probiotic organisms or purified for cell-free sensor applications. However, most natural sensors recognize multiple structurally similar, but functionally diverse, metabolites often found in close proximity. To accurately correlate the concentration of a chemical with biological outcomes, sensors need to differentiate between different chemicals and recognize the relevant chemical with the precise sensitivity.
[0172] Specific sensing is critical for many microbial sensor applications. A variety of protein engineering methods have been demonstrated for optimizing the selectivity and sensitivity of protein and RNA sensors. These approaches include directed evolution and rational design, and computational de novo design. Although each method has advantages, rational engineering can uniquely be performed using both basic knowledge of the protein structure and small library sizes, allowing for rapid construction and screening of libraries. In addition, when the structure of the protein is unknown, conserved or essential residues can also be identified through computational simulations or by aligning the sequence of the sensor with other proteins in the same family. Despite advances in protein engineering, creating ligand-specific sense-and-respond systems remains challenging due to the need to couple subtle protein conformational changes with differential protein-DNA interactions and gene expression control, especially when the target ligands are structurally similar.
[0173] The aromatic amino acids phenylalanine (Phe) and tyrosine (Tyr) are common microbial metabolic engineering products and precursors derived from the same pathway. In addition, chronically elevated levels of structurally similar Phe and Tyr are associated with the distinct disorders phenylketonuria and type 2 tyrosinemia, respectively (see e.g., TABLE 1).
TABLE-US-00005 TABLE 1 Concentrations of phenylalanine, tyrosine, phenylethylamine, and tyramine from different sources. Chemical Source Concentration (mM) Phenylalanine Ileum, healthy 0.30 Plasma, healthy 0.06 Plasma, phenylketonuria 1.1 Serum, healthy 0.06-0.07 Urine, healthy 0.06 Tyrosine Ileum, healthy 0.26 Plasma, healthy 0.07 Plasma, tyrosinemia 1.3 Serum, healthy 0.06-0.08 Urine, healthy 0.04 Phenylethylamine Sauerkraut, safe limit 0.03 Plasma, healthy 0.04 Plasma, schizophrenics 0.10 Tyramine Sauerkraut, safe limit 0.12 Plasma, healthy 0.0007 Plasma, elevated blood pressure >0.001
[0174] Sensors for both metabolites have been generated in different E. coli strains. However, the sensors are based on the wild-type version of the multi-ligand responsive TyrR transcription factor (TF) from E. coli and have limited selectivity and low dynamic ranges. Similarly, the structurally similar amines phenylethylamine (PEA), tyramine (Tyra), and tryptamine (Trypta) are all commonly found in food and in the gut but contribute to distinct biological outcomes (see e.g., TABLE 1). For example, extreme levels of PEA have been associated with a variety of psychological disorders, the presence of Tyra leads to catecholamine release and an increase in blood pressure, and the presence of Trypta causes serotonin release and the stimulation of gastrointestinal motility. Additionally, the presence of PEA, Tyra, and Trypta in food are indicators of microbial contamination, and eating foods with high levels of Tyra can lead to poisoning. Currently, there are no biosensors with high ligand specificity for these chemicals.
[0175] Described herein is the generation of sensors for the aromatic metabolites Phe, Tyr, PEA, and Trypta using Escherichia coli Nissle 1917 (EcN) as a host. To generate sensors for these aromatic metabolites, two sensor systems were identified and engineered: the TyrR (Phe and Tyr) sensor and the TynA-FeaR (aromatic amine) sensor system. The ligand selectivity of the TyrR and TynA-FeaR sensor systems was engineered and their sensitivity was tuned by rationally selecting and individually mutating amino acids in TyrR, TynA, and FeaR. This method of rational protein engineering quickly generates multiple small libraries that can be efficiently screened, making it an attractive approach for specificity engineering. Altogether, the sensors specific for Phe, Tyr, PEA, or Tyra were generated. In engineering FeaR, insights were provided into the uncharacterized structure of FeaR and residues important for ligand binding were identified. Herein are provided sensors with diverse applications in microbial biosensing as well as a generalizable approach to modulating the specificity of ligand-protein binding while maintaining protein-DNA interactions and thus downstream gene expression control.
[0176] Results
[0177] Developing Specific Phenylalanine and Tyrosine Sensors
[0178] Phe and Tyr are structurally similar metabolites utilized throughout the body for many processes as additives in food and animal feed and as precursors for many chemicals and pharmaceuticals (see e.g.,
[0179] First, six E. coli-native promoters were characterized using genomic expression or plasmid-based overexpression of TyrR to identify the best promoter in EcN (see e.g,
[0180] The E274Q TyrR mutation was previously shown to prevent TyrR from forming hexamers, which occurs when TyrR is bound to Tyr. Since hexamerization is required for Tyr-mediated repression of PtyrP, this mutation should render TyrR nonresponsive to Tyr but maintain Phe-dependent induction. When the mutation was inserted into the plasmid-expressed TyrR, Tyr-dependent repression of PtyrP was mitigated and the Tyr-dominating response of the TF was eliminated (see e.g.,
[0181] Next, the Phe-sensitivity of the E274Q mutant was tuned to create sensors that better align with variable physiological concentrations and improve the utility of the sensors for therapeutic and diagnostic applications. Eight amino acid positions with known roles in TyrR-mediated promoter induction were selected (see e.g.,
[0182] TyrR was also engineered for Tyr specificity. Because TyrR binds to Phe and Tyr using two separate binding pockets, the libraries designed to identify less sensitive Phe sensors were used to identify variants completely devoid of Phe-activity, while maintaining Tyr activity (see e.g.,
[0183] Engineering Ligand-Specific Aromatic Amine Sensors
[0184] The body harbors a variety of aromatic amines with diverse neurological functions. Many of these metabolites, including dopamine (DA), PEA, Tyra, and Trypta, can be found in the intestines. Many commensal microbes can convert these amines into aldehydes through monoamine oxidases (see e.g.,
[0185] To develop sensors for aromatic amines, the TynA-FeaR system was first characterized in EcN. A sensor plasmid was constructed, which consisted of constitutively expressed TynA and FeaR from E. coli MG1655, and a reporter plasmid, which consisted of GFP under the control of PtynA (see e.g.,
TABLE-US-00006 TABLE 2 Fitted Hill equation parameters for the specified figures. K.sub.A FIG. Strain Plasmid(s) Ligand F.sub.max F.sub.min n (mM) RMSE 1C sAGR039 pAGR010 Phenylalanine 10500 2900 5.5 0.073 20.9 1C sAGR039 pAGR010 Tyrosine 2470 38.1 1.9 0.026 8.5 1C sAGR039 pAGR010 Phenylalanine 2640 255 1.5 0.032 10.7 and tyrosine 2C, 2D sAGR039 pAGR102 Phenylalanine 12800 982 2 0.042 690 2D sAGR039 pAGR102 Phenylalanine 12700 1060 2.1 0.055 442 and tyrosine 2C, 2E sAGR039 pAGR157 Phenylalanine 7560 814 1.4 0.083 273 2E sAGR039 pAGR157 Phenylalanine 7670 1030 2.1 0.084 178 and tyrosine 2C, 2F sAGR039 pAGR159 Phenylalanine 10700 1310 0.7 0.128 338 2F sAGR039 pAGR159 Phenylalanine 9710 1510 0.8 0.155 212 and tyrosine 2G sAGR039 pAGR473 Tyrosine 3210 279 2.3 0.047 7.7 2G sAGR039 pAGR473 Phenylalanine 3110 3.9 2 0.034 10.2 and Tyrosine 3D sAGR039 pCX008, Dopamine 881 0.1 3 0.068 5.2 pHJY022 3D, S15a sAGR039 pCX008, Phenylethylamine 1430 0 3.9 0.087 5.9 pHJY022 3D sAGR039 pCX008, Tyramine 1470 4.4 5 0.077 9.3 pHJY022 3D sAGR039 pCX008, Tryptamine 268 1.3 4.3 0.085 9.9 pHJY022 4C, 22B sAGR039 pCX008, Phenylethylamine 790 5.4 1.4 1.8 5.2 pCX014 4D sAGR039 pCX008, Phenylethylamine 84 0 0.6 0.012 8.7 pCX015 4D sAGR039 pCX008, Tyramine 226 0 0.9 0.023 25.4 pCX015 5C sAGR039 pCX008, Phenylethylamine 585 4.8 2.7 0.137 4 pCX024 6D sAGR039 pCX008, Phenylethylamine 1004 5.8 2.1 0.012 32.3 pCX068 6E sAGR039 pCX008, Tyramine 158 1.5 12 0.012 21.7 pCX073 10A sAGR039 pAGR160 Phenylalanine 10800 1120 2.5 0.046 790 10B sAGR039 pAGR155 Phenylalanine 12900 5570 2.2 0.045 1090 10C sAGR039 pAGR156 Phenylalanine 12400 1430 2.4 0.037 680 10D sAGR039 pAGR158 Phenylalanine 11800 2150 1 0.11 397 14 sAGR039 pCX008, Dopamine 258 4.4 1.5 0.028 5.2 pHJY022 14 sAGR039 pCX008, Phenylethylamine 315 10.4 2 0.042 5.9 pHJY022 14 sAGR039 pCX008, Tyramine 311 10.6 1.6 0.037 9.3 pHJY022 14 sAGR039 pCX008, Tryptamine 57 5.9 3.1 0.045 9.9 pHJY022 17A sAGR039 pCX008, Phenylethylamine 9280 1.1 1.2 33 3.2 pCX014 17B sAGR039 pCX008, Phenylethylamine 118 0 0.4 1.64 8.6 pCX015 17B sAGR039 pCX008, Tyramine 152 0 0.8 0.041 16.7 pCX015 18A sAGR039 pCX008, Phenylethylamine 18 0.8 36 0.727 0.8 pCX010 18B, 22B sAGR039 pCX008, Phenylethylamine 4012 3.4 1.3 43 3.9 pCX010 22A sAGR039 pCX008, Phenylethylamine 1010 18 4.3 0.046 2.8 pCX016 23A sAGR039 pCX008, Phenylethylamine 462 3.4 1.2 3.33 5.1 pCX032 25B, 22B sAGR039 pCX008, Phenylethylamine 475 5.9 1.5 1.34 9.1 pCX032 24 sAGR039 pCX008, Phenylethylamine 289 5.1 2.6 0.074 5.8 pCX024 25A sAGR039 pCX008, Phenylethylamine 14 0.4 8.4 0.014 1 pCX025 25B sAGR039 pCX008, Phenylethylamine 112 1.1 20.3 0.052 4.3 pCX025 28A sAGR039 pCX008, Tyramine 55 0.4 8.4 0.011 13.7 pCX083 28B sAGR039 pCX008, Tyramine 78 1.8 1.6 0.008 13.9 pCX099 28C sAGR039 pCX008, Tyramine 127 1.5 9.8 0.027 19.7 pCX116 28D sAGR039 pCX008, Tyramine 148 0.9 9.7 0.011 13.6 pCX117
[0186] Due to their superior induction and roles in numerous processes, PEA and Tyra were selected as targets for engineering selectivity in the TynA-FeaR system. This system presents an interesting opportunity to optimize ligand selectivity using two different knobs of control. Either TynA or FeaR can be engineered for selectivity. However, the protein structure and mode of ligand binding of FeaR have not been elucidated. In contrast, the structure of TynA has been characterized. Thus, improving the amine selectivity of TynA was focused on first. Several residues in TynA have been identified as catalytically essential, including D413 and Y496 (see e.g.,
[0187] To quickly screen variants from the libraries for a response to each ligand, a growth-based assay was developed by incorporating the carbenicillin resistance gene, bla, and the sucrose counterselection gene, sacB, onto the reporter plasmid under the control of PtynA (see e.g.,
[0188] Two PEA-specific variants were successfully identified (see e.g.,
[0189] Two variants with improved selectivity to Tyra were also identified (see e.g.,
[0190] Characterization and Engineering of FeaR
[0191] Intrigued by the appearance of the A81T FeaR mutation in the G494S* sensor, next the importance of the 81st residue for ligand binding was explored. A protein motif search indicated that the position 12-185 of FeaR is a ligand-binding domain of the AraC-like TF, and the position 218-298 is its DNA-binding domain with a helix-turn-helix motif at positions 258-298. Since no experimentally derived structures of FeaR are known to exist, computational simulations were applied based on comparative modeling to predict the structure. The algorithm utilized the moderate homology of FeaR to CuxR, another AraC-like TF with a previously solved structure, to comparatively predict the structure of FeaR. Visualization of the predicted structure revealed that A81 is part of a solvent-accessible beta-barrel, and the alanine side chain is oriented toward the interior of the barrel (see e.g.,
[0192] To further elucidate the role of the A81T mutation in ligand selectivity, the A81T mutation was inserted into the FeaR-TynA sensor plasmid with wild-type TynA. The mutation had an insignificant impact on the maximum response of the sensor to PEA and Tyra but significantly reduced the responses to DA and Trypta (see e.g.,
[0193] Noting the PEA selectivity given by the A81T mutation and the smallest size of the PEA-aldehyde (no OH group), followed by that of Tyra—(1 OH), DA—(2 OHs), and Trypta—(additional 5-membered ring) aldehyde (see e.g.,
TABLE-US-00007 TABLE 3 Ligand specificity of FeaR with different residues in the 81 position. Residues are listed in order of increasing size. Amino acid FeaR A81 Size (×10.sup.−3 family variant nm.sup.3) HPI Phenotype Selectivity Hydrophobic Glycine (G) 59.9 −0.4 PEA/Tyra PEA ≈ Tyra > selective DA Alanine (A) 87.8 1.8 Non specific PEA ≈ Tyra > DA > Trypta Proline (P)* 123.3 −1.6 PEA specific PEA only Valine (V) 138.8 4.2 PEA/Tyra PEA > Tyra selective No DA or Trypta activity Methionine (M) 165.2 1.9 PEA/Tyra PEA ≈ Tyra > selective DA No Trypta activity Isoleucine (I) 166.1 4.5 PEA specific PEA only Leucine (L) 168 3.8 PEA specific PEA only Phenylalanine (F)** 189.7 2.8 No activity No activity Tyrosine (Y)** 191.2 −1.3 No activity No activity Tryptophan (W)** 227.9 −0.9 No activity No activity Polar Serine (S) 91.7 −0.8 PEA/Tyra PEA > Tyra > selective DA > Trypta Cysteine (C) 105.4 2.5 Non specific PEA > Tyra > DA > Trypta Threonine (T) 118.3 0.7 PEA/Tyra PEA > Tyra > selective DA > Trypta Asparagine (N) 120.1 −3.5 PEA specific PEA only Glutamine (Q) 145.1 −3.5 No activity No activity Charged Histidine (H) 156.3 −3.2 No activity No activity (positive) Lysine (K) 172.7 −3.9 No activity No activity Arginine (R) 188.2 −4.5 No activity No activity Charged Aspartic Acid (D) 115.4 −3.5 No activity No activity (negative) Glutamic Acid (E) 140.9 −3.5 No activity No activity *P is more structurally rigid than the other amino acids. **F, Y, and W are aromatic amino acids. HPI, hydropathy index.
[0194] FeaR recognized Tyra-aldehyde at a range of size and hydropathy index values (see e.g.,
[0195] Structural simulations of the wild-type (promiscuous), A81T (Tyra- and PEA-responsive), A81L (PEA-specific), and A81H (non-functional) variants were performed to understand how the mutations may alter the structure and thus ligand binding of FeaR. Residues of increasing size (A<T<L) protruded further into and occupied more space in the ligand-binding pocket, which is consistent with the observed size effect. All three mutations also shifted the positions of several side chains within the beta-barrel relative to the wild-type structure (see e.g.,
[0196] Given their significant rotations, it was hypothesized that residues M83, L108, and W110 may also play important roles in ligand binding, potentially by making contacts with alternative functional groups in the ligand. It was hypothesized that mutagenizing these residues may reveal a sensor for DA, Tyra, or Trypta. Each residue was individually mutated and multiple ligand-specific sensors for PEA or Tyra were identified (see e.g.,
[0197] To further confirm the specificity of the best performing sensors, the response of each to the four amines and the four respective carboxylic acids, 3,4-dihydroxyphenylacetic acid (DOPAC), phenylacetic acid (PAA), 4-hydroxyphenylacetic acid (HPAA), and indole-3-acetic acid (IAA) was tested in minimal medium with casamino acids and LB medium (see e.g.,
[0198] Together, this work provides insights into the structure and activity of FeaR. In addition, by engineering FeaR, the best performing PEA- and Tyra-specific sensors are provided for future applications. Future work could include random approaches of protein engineering such as error-prone PCR-based directed evolution to develop additional ligand-specific sensors for the larger DA and Trypta amines. In addition, further exploration of the relevant regulatory pathways is required to uncouple the activity of the sensors with resource availability.
[0199] Discussion
[0200] The ligand-specific sensors developed here have the potential for diverse applications, including (1) monitoring food quality, (2) diagnosing or treating metabolic, digestive, and neurological disorders in probiotics or ex vivo wearable, paper-based and cell-free systems, and (3) dynamically regulating enzymatic pathways for microbial metabolic engineering. The high degree of ligand specificity shown by the engineered sensors allows them to effectively differentiate between diverse structurally similar aromatic metabolites. Demonstrated herein is an efficient and effective method of rational protein engineering by individually performing saturation mutagenesis on logically selected amino acid residues. The generalizability of this method is shown by applying it to three protein systems, including both enzymes and TFs. The simplicity of the approach and small library sizes make it an attractive first step in sensor engineering. Although this protein engineering method requires basic structural knowledge of the protein of interest, the scope of the required information is less than fully computation-based engineering approaches. However, also shown herein is how protein simulations can be used to identify important residues from uncharacterized proteins.
[0201] Protein engineering for specific ligand-protein interactions has been extensively performed, especially for facilitating chemical drug screening. However, coupling ligand-protein binding with an output response has remained challenging. Current approaches include designing proteins that fluoresce upon ligand binding or employing ligand-binding TFs. While the former can be used to develop sensitive sensors, the latter can generate sensors as well as controllers for downstream functions such as gene expression. Because this TF-based system requires the maintenance or engineering of DNA-protein interaction in addition to engineering ligand-protein binding, developing ligand-specific sensors using this system has been challenging, especially when the target ligands are structurally similar. To address this challenging issue, two approaches were demonstrated: optimizing ligand-TF binding specificity and sensitivity by leveraging differential multimerization patterns of TyrR without affecting DNA-TF binding interactions (see e.g.,
[0202] The feaR-tynA system represents a unique way to develop sensors using two different control knobs. As shown in
[0203] Altogether, the specific microbial sensors for Phe, Tyr, PEA, and Tyra were generated. This work provides ligand-specific sensors that can be applied to create probiotics for diverse applications. In addition, the generalizable protein engineering techniques demonstrated here can be used to quickly and effectively engineer enzymes and TFs for ligand specificity and sensitivity. Although sensors were specifically developed with potential applications primarily in medicine and food quality, these enzymes and sensors can be similarly engineered to produce fuels, pharmaceuticals, and commodity chemicals in response to the levels of metabolites or the products in a dynamic way. This work represents a considerable achievement in the challenging goal of engineering ligand-specific sense-and-respond systems for medically relevant chemicals through coupling protein conformational changes caused by ligand binding with DNA interactions.
[0204] Methods
[0205] Plasmids, Strains, and Reagents
[0206] All plasmids, strains, and genetic parts used in this study are summarized in TABLE 4, TABLE 5, and TABLE 6, respectively.
[0207] It is noted that TrpR and Ptrp constructs are either WT sequences form E. coli or from the following paper: Ellefson, J. W., Ledbetter, M. P. & Ellington, A. D. Directed evolution of a synthetic phylogeny of programmable Trp repressors. Nat Chem Biol 14, 361-367 (2018). https://doi.org/10.1038/s41589-018-0006-7.
TABLE-US-00008 TABLE 4 Plasmids used in this work. Plasmid Genetic Antibiotic Name Parts Origin Resistance Source Recombineering pMP11 Pcon-cas9 + pBAD-λ oriR101 Ampicillin Mehrer et al. Red genes + Ptet- sgRNA-pBR322ori pgRNAcm Constitutive sgRNA pBR322 Chloramphenicol Mehrer et al. pMBA016 pgRNA-trpR pBR322 Chloramphenicol This work TrpR-based tryptophan, 5-hydroxytryptophan, and tryptamine sensors pMBA014 Ptrp-gfp ColE1 Chloramphenicol This work; Ptrp from Ellefson et al. pMBA035 P17-trpR p15A Spectinomycin This work pMBA077 Ptrp(O.sub.D)-gfp ColE1 Chloramphenicol This work pMBA094 Ptrp(O.sub.1)-gfp ColE1 Chloramphenicol This work pMBA103 P17-trpR(O.sub.D) p15A Spectinomycin This work pMBA173 P17-trpR(O.sub.1) p15A Spectinomycin This work TyrR-based phenylalanine and tyrosine sensors pAGR001 PtyrR-gfp ColE1 Chloramphenicol This work pAGR002 Pmtr-gfp ColE1 Chloramphenicol This work pAGR003 PtyrB-gfp ColE1 Chloramphenicol This work pAGR009 ParoF-gfp ColE1 Chloramphenicol This work pAGR010 PtyrP-gfp ColE1 Chloramphenicol This work pAGR011 ParoP-gfp ColE1 Chloramphenicol This work pAGR023 PtyrP-gfp + PtyrR-tyrR ColE1 Chloramphenicol This work; derived from pAGR010 pAGR031 ParoF*-gfp + PtyrR-tyrR ColE1 Chloramphenicol This work; derived from pAGR009 pAGR032 ParoP*-gfp + PtyrR-tyrR ColE1 Chloramphenicol This work; derived from pAGR011 pAGR102 PtyrP-gfp + PtyrR- ColE1 Chloramphenicol This tyrR_E274Q work; derived from pAGR023 pAGR155 PtyrP-gfp + PtyrR- ColE1 Chloramphenicol This tyrR_E274Q_L11G work; derived from pAGR102 pAGR156 PtyrP-gfp + PtyrR- ColE1 Chloramphenicol This tyrR_E274Q_T14G work; derived from pAGR102 pAGR157 PtyrP-gfp + PtyrR- ColE1 Chloramphenicol This tyrR_E274Q_T14V work; derived from pAGR102 pAGR158 PtyrP-gfp + PtyrR- ColE1 Chloramphenicol This tyrR_E274Q_D103H work; derived from pAGR102 pAGR159 PtyrP-gfp + PtyrR- ColE1 Chloramphenicol This tyrR_E274Q_D103S work; derived from pAGR102 pAGR160 PtyrP-gfp + PtyrR- ColE1 Chloramphenicol This tyrR_E274Q_C7V work; derived from pAGR102 pAGR473 PtyrP-gfp + PtyrR- ColE1 Chloramphenicol This tyrR_R10F work; derived from pAGR023 pAGR475 PtyrP-gfp + PtyrR- ColE1 Chloramphenicol This tyrR_L11G work; derived from pAGR023 pAGR476 PtyrP-gfp + PtyrR- ColE1 Chloramphenicol This tyrR_G12T work; derived from pAGR023 pAGR477 PtyrP-gfp + PtyrR- ColE1 Chloramphenicol This tyrR_L13G work; derived from pAGR023 pAGR479 PtyrP-gfp + PtyrR- ColE1 Chloramphenicol This tyrR_T14K work; derived from pAGR023 TynA-FeaR-based amine sensors pHJY022 Pcon-feaR + Pcon-tynA pSC101 Kanamycin This work pHJY023 Pcon-feaR pSC101 Kanamycin This work pHJY028 PtynA-gfp p15A Spectinomycin This work pCX008 PtynA-gfp-bla-sacB p15A Spectinomycin This work pCX010 Pcon-feaR + Pcon- pSC101 Kanamycin This tynA_G494S work; derived from pHJY022 pCX014 Pcon-feaR_A81T + pSC101 Kanamycin This Pcon-tynA_G494S work; derived from pHJY022 pCX015 Pcon-feaR + Pcon- pSC101 Kanamycin This tynA_G415H work; derived from pHJY022 pCX019 Pcon-feaR + Pcon- pSC101 Kanamycin This tynA_S414M work; derived from pHJY022 pCX020 Pcon-feaR + Pcon- pSC101 Kanamycin This tynA_G415Y work; derived from pHJY022 pCX016 Pcon-feaR_A81T + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX017 Pcon-feaR_A81T + pSC101 Kanamycin This Pcon-tynA_S414M work; derived from pCX019 pCX018 Pcon-feaR_A81T + pSC101 Kanamycin This Pcon-tynA_G415H work; derived from pCX015 pCX024 Pcon-feaR_A81L + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX025 Pcon-feaR_A81P + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX032 Pcon-feaR_A81S + pSC101 Kanamycin This Pcon-tynA_G494S work; derived from pCX010 pCX033 Pcon-feaR_A81S + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX035 Pcon-feaR_A81V + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX036 Pcon-feaR_A81E + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX037 Pcon-feaR_A81M + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX039 Pcon-feaR_A81G + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX040 Pcon-feaR_A81I + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX041 Pcon-feaR_A81F + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX042 Pcon-feaR_A81Y + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX043 Pcon-feaR_A81W + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX034 Pcon-feaR_A81C + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX044 Pcon-feaR_A81N + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX045 Pcon-feaR_A81Q + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX046 Pcon-feaR_A81K + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX047 Pcon-feaR_A81H + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX048 Pcon-feaR_A81R + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX049 Pcon-feaR_A81D + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX075 Pcon-feaR_M83G + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX072 Pcon-feaR_M83A + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX070 Pcon-feaR_M83P + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX076 Pcon-feaR_M83V + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX077 Pcon-feaR_M83I + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX078 Pcon-feaR_M83L + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX074 Pcon-feaR_M83F + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX073 Pcon-feaR_M83Y + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX080 Pcon-feaR_M83W + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX081 Pcon-feaR_M83S + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX082 Pcon-feaR_M83C + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX071 Pcon-feaR_M83T + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX068 Pcon-feaR_M83N + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX083 Pcon-feaR_M83Q + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX084 Pcon-feaR_M83H + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX085 Pcon-feaR_M83K + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX086 Pcon-feaR_M83R + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX087 Pcon-feaR_M83D + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX069 Pcon-feaR_M83E + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX088 Pcon-feaR_L108G + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX089 Pcon-feaR_L108A + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX090 Pcon-feaR_L108P + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX091 Pcon-feaR_L108V + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX092 Pcon-feaR_L108M + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX093 Pcon-feaR_L108I + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX094 Pcon-feaR_L108F + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX095 Pcon-feaR_L108Y + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX096 Pcon-feaR_L108W + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX097 Pcon-feaR_L108S + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX098 Pcon-feaR_L108C + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX099 Pcon-feaR_L108T + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX100 Pcon-feaR_L108N + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX101 Pcon-feaR_L108Q + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX102 Pcon-feaR_L108H + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX103 Pcon-feaR_L108K + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX104 Pcon-feaR_L108R + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX105 Pcon-feaR_L108D + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX106 Pcon-feaR_L108E + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX107 Pcon-feaR_W110G + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX108 Pcon-feaR_W110A + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX109 Pcon-feaR_W110P + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX110 Pcon-feaR_W110V + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX111 Pcon-feaR_W110M + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX112 Pcon-feaR_W110I + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX113 Pcon-feaR_W110L + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX114 Pcon-feaR_W110F + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX115 Pcon-feaR_W110Y + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX116 Pcon-feaR_W110S + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX117 Pcon-feaR_W110C + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX118 Pcon-feaR_W110T + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX119 Pcon-feaR_W110N + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX120 Pcon-feaR_W110Q + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX121 Pcon-feaR_W110H + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX122 Pcon-feaR_W110K+ pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX123 Pcon-feaR_W110R + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX124 Pcon-feaR_W110D + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022 pCX125 Pcon-feaR_W110E + pSC101 Kanamycin This Pcon-tynA work; derived from pHJY022
TABLE-US-00009 TABLE 5 E. coli strains used in this work. Name Strain Source sAGR004 DH10B Invitrogen sAGR039 Nissle 1917 (plasmid DSMZ free)
TABLE-US-00010 TABLE 6 Genetic parts used in this work. SEQ ID NO: Part Nucleotide Sequence General Parts 1 gfpmut3 with RBS aagtcggtgacagataacaggagtaagtaATGagtaaaggaga agaacttttcactggagttgtcccaattcttgttgaattagatggtgatgtt aatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaac atacggaaaacttacccttaaatttatttgcactactggaaaactacct gttccatggccaacacttgtcactactttgacttatggtgttcaatgcttttc aagatacccagatcatatgaaacggcatgactttttcaagagtgccat gcccgaaggttatgtacaggaaagaactatatttttcaaagatgacg ggaactataagacacgtgctgaagtcaagtttgaaggtgatacactt gttaatagaatcgagttaaaaggtattgattttaaagaagatggaaac attcttggacacaagttggaatacaactataactcacacaatgtatac atcatggcagacaaacaaaagaatggaatcaaagttaacttcaaa attagacacaacattgaagatggaagcgttcaactagcagaccatta tcaacaaaatactccaattggcgatggccctgtccttttaccagacaa ccattacctgtccacacaatctgccctttcgaaagatcccaacgaaa agagagaccacatggtccttcttgagtttgtaacagctgctgggattac acatggcatggatgaactatacaaaaggcctgcagcaaacgacga aaactacgcttaagtagcttaa 2 BBa_J23117 (P17) ttgacagctagctcagtcctagggattgtgctagc TyrR Sensor 3 PtyrR ccagcgaaaaataatgcaatatcgggtgctgaccggatatctttacg ccgaagtgcccgtttttccgtctttgtgtcaatgattgttgacagaaacct tcctgctatccaaatagtgtcatatcatcatatta 4 Pmtr cgtcgtttcggtggtgatgcgtaatcatcgctgaacagcgaacacaat ctgtaaaataatatatacagccccgatttttaccatcggggctttttttctg tcttttgtactcgtgtactggtacagtgcaatgc 5 PtyrB with start tctgttgctaattgccgttcgctcctgaacatccactcgatcttcgccttctt codon ccggtttattgtgttttaaccacctgcccgtaaacctggagaaccatcg cGTGtttcaaaaagttgacgcc 6 ParoF attgttttcaaagggagtgtaaatttatctatacagaggtaagggttgaa agcgcgactaaattgcctgtgtaaataaaaatgtacgaaatatggatt gaaaactttactttatgtgttatcgttacgtc 7 PtyrP cgtccctcctcaaaaaaagcctagcgtagcgattgccgcttatgaag actttgcgccagcgcaggactgaatgctttttattgtacatttatatttaca ccatatgtaacgtcggtttgacgaagcagccgttatgccttaacctgcg ccgcag 8 ParoP aagcaactcatcttcaaccatgcataaagcgggtgcattcgctgccg cataccattattcttgatctgacggaagtctttttgtaacaattcaaacttc tttgatgtaaacaaattaatacaacaaacggaattgcaaacttacaca cgc 9 ParoF* attgttttcaaagggagtgtaaatttatctatacagaggtaagggttgaa agcgcgactaaattgcctgtgtaaataaaaatgtacgaaatatggatt gaaaactttactttatgaggtataatgctagc 10 ParoP* aagcaactcatcttcaaccatgcataaagcgggtgcattcgctgccg cataccattattcttgacagctagctcagtcctaggtataatgctagcttc tttgatgtaaacaaattaatacaacaaacggaattgcaaacttacaca cgc 11 tyrR [with RBS] [attgttcttttttcaggtgaaggttccc]ATGcgtctggaagtcttttgtg aagaccgactcggtctgacccgcgaattactcgatctactcgtgctaa gaggcattgatttacgcggtattgagattgatcccattgggcgaatcta cctcaattttgctgaactggagtttgagagtttcagcagtctgatggccg aaatacgccgtattgcgggtgttaccgatgtgcgtactgtcccgtggat gccttccgaacgtgagcatctggcgttgagcgcgttactggaggcgtt gcctgaacctgtgctctctgtcgatatgaaaagcaaagtggatatggc gaacccggcgagctgtcagctttttgggcaaaaattggatcgcctgc gcaaccataccgccgcacaattgattaacggctttaattttttacgttgg ctggaaagcgaaccgcaagattcgcataacgagcatgtcgttattaa tgggcagaatttcctgatggagattacgcctgtttatcttcaggatgaaa atgatcaacacgtcctgaccggtgcggtggtgatgttgcgatcaacg attcgtatgggccgccagttgcaaaatgtcgccgcccaggacgtcag cgccttcagtcaaattgtcgccgtcagcccgaaaatgaagcatgttgt cgaacaggcgcagaaactggcgatgctaagcgcgccgctgctgatt acgggtgacacaggtacaggtaaagatctctttgcctacgcctgccat caggcaagccccagagcgggcaaaccttacctggcgctgaactgt gcgtctataccggaagatgcggtcgagagtgaactgtttggtcatgct ccggaagggaagaaaggattctttgagcaggcgaacggtggttcg gtgctgttggatgaaataggggaaatgtcaccacggatgcaggcga aattactgcgtttccttaatgatggcactttccgtcgggttggcgaagac catgaggtgcatgtcgatgtgcgggtgatttgcgctacgcagaagaat ctggtcgaactggtgcaaaaaggcatgttccgtgaagatctctattatc gtctgaacgtgttgacgctcaatctgccgccgctacgtgactgtccgc aggacatcatgccgttaactgagctgttcgtcgcccgctttgccgacg agcagggcgtgccgcgtccgaaactggccgctgacctgaatactgt acttacgcgttatgcgtggccgggaaatgtgcggcagttaaagaacg ctatctatcgcgcactgacacaactggacggttatgagctgcgtccac aggatattttgttgccggattatgacgccgcaacggtagccgtgggcg aagatgcgatggaaggttcgctggacgaaatcaccagccgttttgaa cgctcggtattaacccagctttatcgcaattatcccagcacgcgcaaa ctggcaaaacgtctcggcgtttcacataccgcgattgccaataagttg cgggaatatggtctgagtcagaagaagaacgaagagtaa 12 tyrR_E274Q ATGcgtctggaagtcttttgtgaagaccgactcggtctgacccgcga attactcgatctactcgtgctaagaggcattgatttacgcggtattgaga ttgatcccattgggcgaatctacctcaattttgctgaactggagtttgag agtttcagcagtctgatggccgaaatacgccgtattgcgggtgttacc gatgtgcgtactgtcccgtggatgccttccgaacgtgagcatctggcgt tgagcgcgttactggaggcgttgcctgaacctgtgctctctgtcgatat gaaaagcaaagtggatatggcgaacccggcgagctgtcagctttttg ggcaaaaattggatcgcctgcgcaaccataccgccgcacaattgatt aacggctttaattttttacgttggctggaaagcgaaccgcaagattcgc ataacgagcatgtcgttattaatgggcagaatttcctgatggagattac gcctgtttatcttcaggatgaaaatgatcaacacgtcctgaccggtgc ggtggtgatgttgcgatcaacgattcgtatgggccgccagttgcaaaa tgtcgccgcccaggacgtcagcgccttcagtcaaattgtcgccgtca gcccgaaaatgaagcatgttgtcgaacaggcgcagaaactggcga tgctaagcgcgccgctgctgattacgggtgacacaggtacaggtaa agatctctttgcctacgcctgccatcaggcaagccccagagcgggc aaaccttacctggcgctgaactgtgcgtctataccggaagatgcggtc gagagtcaactgtttggtcatgctccggaagggaagaaaggattcttt gagcaggcgaacggtggttcggtgctgttggatgaaataggggaaa tgtcaccacggatgcaggcgaaattactgcgtttccttaatgatggca ctttccgtcgggttggcgaagaccatgaggtgcatgtcgatgtgcggg tgatttgcgctacgcagaagaatctggtcgaactggtgcaaaaaggc atgttccgtgaagatctctattatcgtctgaacgtgttgacgctcaatctg ccgccgctacgtgactgtccgcaggacatcatgccgttaactgagct gttcgtcgcccgctttgccgacgagcagggcgtgccgcgtccgaaa ctggccgctgacctgaatactgtacttacgcgttatgcgtggccggga aatgtgcggcagttaaagaacgctatctatcgcgcactgacacaact ggacggttatgagctgcgtccacaggatattttgttgccggattatgac gccgcaacggtagccgtgggcgaagatgcgatggaaggttcgctg gacgaaatcaccagccgttttgaacgctcggtattaacccagctttatc gcaattatcccagcacgcgcaaactggcaaaacgtctcggcgtttca cataccgcgattgccaataagttgcgggaatatggtctgagtcagaa gaagaacgaagagtaa 13 tyrR_E274Q_L11G ATGcgtctggaagtcttttgtgaagaccgagggggtctgacccgcg aattactcgatctactcgtgctaagaggcattgatttacgcggtattgag attgatcccattgggcgaatctacctcaattttgctgaactggagtttga gagtttcagcagtctgatggccgaaatacgccgtattgcgggtgttac cgatgtgcgtactgtcccgtggatgccttccgaacgtgagcatctggc gttgagcgcgttactggaggcgttgcctgaacctgtgctctctgtcgat atgaaaagcaaagtggatatggcgaacccggcgagctgtcagctttt tgggcaaaaattggatcgcctgcgcaaccataccgccgcacaattg attaacggctttaattttttacgttggctggaaagcgaaccgcaagattc gcataacgagcatgtcgttattaatgggcagaatttcctgatggagatt acgcctgtttatcttcaggatgaaaatgatcaacacgtcctgaccggt gcggtggtgatgttgcgatcaacgattcgtatgggccgccagttgcaa aatgtcgccgcccaggacgtcagcgccttcagtcaaattgtcgccgt cagcccgaaaatgaagcatgttgtcgaacaggcgcagaaactggc gatgctaagcgcgccgctgctgattacgggtgacacaggtacaggt aaagatctctttgcctacgcctgccatcaggcaagccccagagcgg gcaaaccttacctggcgctgaactgtgcgtctataccggaagatgcg gtcgagagtcaactgtttggtcatgctccggaagggaagaaaggatt ctttgagcaggcgaacggtggttcggtgctgttggatgaaatagggg aaatgtcaccacggatgcaggcgaaattactgcgtttccttaatgatg gcactttccgtcgggttggcgaagaccatgaggtgcatgtcgatgtgc gggtgatttgcgctacgcagaagaatctggtcgaactggtgcaaaaa ggcatgttccgtgaagatctctattatcgtctgaacgtgttgacgctcaa tctgccgccgctacgtgactgtccgcaggacatcatgccgttaactga gctgttcgtcgcccgctttgccgacgagcagggcgtgccgcgtccga aactggccgctgacctgaatactgtacttacgcgttatgcgtggccgg gaaatgtgcggcagttaaagaacgctatctatcgcgcactgacaca actggacggttatgagctgcgtccacaggatattttgttgccggattatg acgccgcaacggtagccgtgggcgaagatgcgatggaaggttcgc tggacgaaatcaccagccgttttgaacgctcggtattaacccagcttta tcgcaattatcccagcacgcgcaaactggcaaaacgtctcggcgttt cacataccgcgattgccaataagttgcgggaatatggtctgagtcag aagaagaacgaagagtaa 14 tyrR_E274Q_T14G ATGcgtctggaagtcttttgtgaagaccgactcggtctgacccgcga attactcgatctactcgtgctaagaggcattgatttacgcggtattgaga ttgatcccattgggcgaatctacctcaattttgctgaactggagtttgag agtttcagcagtctgatggccgaaatacgccgtattgcgggtgttacc gatgtgcgtactgtcccgtggatgccttccgaacgtgagcatctggcgt tgagcgcgttactggaggcgttgcctgaacctgtgctctctgtcgatat gaaaagcaaagtggatatggcgaacccggcgagctgtcagctttttg ggcaaaaattggatcgcctgcgcaaccataccgccgcacaattgatt aacggctttaattttttacgttggctggaaagcgaaccgcaagattcgc ataacgagcatgtcgttattaatgggcagaatttcctgatggagattac gcctgtttatcttcaggatgaaaatgatcaacacgtcctgaccggtgc ggtggtgatgttgcgatcaacgattcgtatgggccgccagttgcaaaa tgtcgccgcccaggacgtcagcgccttcagtcaaattgtcgccgtca gcccgaaaatgaagcatgttgtcgaacaggcgcagaaactggcga tgctaagcgcgccgctgctgattacgggtgacacaggtacaggtaa agatctctttgcctacgcctgccatcaggcaagccccagagcgggc aaaccttacctggcgctgaactgtgcgtctataccggaagatgcggtc gagagtcaactgtttggtcatgctccggaagggaagaaaggattcttt gagcaggcgaacggtggttcggtgctgttggatgaaataggggaaa tgtcaccacggatgcaggcgaaattactgcgtttccttaatgatggca ctttccgtcgggttggcgaagaccatgaggtgcatgtcgatgtgcggg tgatttgcgctacgcagaagaatctggtcgaactggtgcaaaaaggc atgttccgtgaagatctctattatcgtctgaacgtgttgacgctcaatctg ccgccgctacgtgactgtccgcaggacatcatgccgttaactgagct gttcgtcgcccgctttgccgacgagcagggcgtgccgcgtccgaaa ctggccgctgacctgaatactgtacttacgcgttatgcgtggccggga aatgtgcggcagttaaagaacgctatctatcgcgcactgacacaact ggacggttatgagctgcgtccacaggatattttgttgccggattatgac gccgcaacggtagccgtgggcgaagatgcgatggaaggttcgctg gacgaaatcaccagccgttttgaacgctcggtattaacccagctttatc gcaattatcccagcacgcgcaaactggcaaaacgtctcggcgtttca cataccgcgattgccaataagttgcgggaatatggtctgagtcagaa gaagaacgaagagtaa 15 tyrR_E274Q_T14V ATGcgtctggaagtcttttgtgaagaccgactcggtctggggcgcga attactcgatctactcgtgctaagaggcattgatttacgcggtattgaga ttgatcccattgggcgaatctacctcaattttgctgaactggagtttgag agtttcagcagtctgatggccgaaatacgccgtattgcgggtgttacc gatgtgcgtactgtcccgtggatgccttccgaacgtgagcatctggcgt tgagcgcgttactggaggcgttgcctgaacctgtgctctctgtcgatat gaaaagcaaagtggatatggcgaacccggcgagctgtcagctttttg ggcaaaaattggatcgcctgcgcaaccataccgccgcacaattgatt aacggctttaattttttacgttggctggaaagcgaaccgcaagattcgc ataacgagcatgtcgttattaatgggcagaatttcctgatggagattac gcctgtttatcttcaggatgaaaatgatcaacacgtcctgaccggtgc ggtggtgatgttgcgatcaacgattcgtatgggccgccagttgcaaaa tgtcgccgcccaggacgtcagcgccttcagtcaaattgtcgccgtca gcccgaaaatgaagcatgttgtcgaacaggcgcagaaactggcga tgctaagcgcgccgctgctgattacgggtgacacaggtacaggtaa agatctctttgcctacgcctgccatcaggcaagccccagagcgggc aaaccttacctggcgctgaactgtgcgtctataccggaagatgcggtc gagagtcaactgtttggtcatgctccggaagggaagaaaggattcttt gagcaggcgaacggtggttcggtgctgttggatgaaataggggaaa tgtcaccacggatgcaggcgaaattactgcgtttccttaatgatggca ctttccgtcgggttggcgaagaccatgaggtgcatgtcgatgtgcggg tgatttgcgctacgcagaagaatctggtcgaactggtgcaaaaaggc atgttccgtgaagatctctattatcgtctgaacgtgttgacgctcaatctg ccgccgctacgtgactgtccgcaggacatcatgccgttaactgagct gttcgtcgcccgctttgccgacgagcagggcgtgccgcgtccgaaa ctggccgctgacctgaatactgtacttacgcgttatgcgtggccggga aatgtgcggcagttaaagaacgctatctatcgcgcactgacacaact ggacggttatgagctgcgtccacaggatattttgttgccggattatgac gccgcaacggtagccgtgggcgaagatgcgatggaaggttcgctg gacgaaatcaccagccgttttgaacgctcggtattaacccagctttatc gcaattatcccagcacgcgcaaactggcaaaacgtctcggcgtttca cataccgcgattgccaataagttgcgggaatatggtctgagtcagaa gaagaacgaagagtaa 16 tyrR_E274Q_D103H ATGcgtctggaagtcttttgtgaagaccgactcggtctgacccgcga attactcgatctactcgtgctaagaggcattgatttacgcggtattgaga ttgatcccattgggcgaatctacctcaattttgctgaactggagtttgag agtttcagcagtctgatggccgaaatacgccgtattgcgggtgttacc gatgtgcgtactgtcccgtggatgccttccgaacgtgagcatctggcgt tgagcgcgttactggaggcgttgcctgaacctgtgctctctgtcgatat gaaaagcaaagtgcacatggcgaacccggcgagctgtcagcttttt gggcaaaaattggatcgcctgcgcaaccataccgccgcacaattga ttaacggctttaattttttacgttggctggaaagcgaaccgcaagattcg cataacgagcatgtcgttattaatgggcagaatttcctgatggagatta cgcctgtttatcttcaggatgaaaatgatcaacacgtcctgaccggtgc ggtggtgatgttgcgatcaacgattcgtatgggccgccagttgcaaaa tgtcgccgcccaggacgtcagcgccttcagtcaaattgtcgccgtca gcccgaaaatgaagcatgttgtcgaacaggcgcagaaactggcga tgctaagcgcgccgctgctgattacgggtgacacaggtacaggtaa agatctctttgcctacgcctgccatcaggcaagccccagagcgggc aaaccttacctggcgctgaactgtgcgtctataccggaagatgcggtc gagagtcaactgtttggtcatgctccggaagggaagaaaggattcttt gagcaggcgaacggtggttcggtgctgttggatgaaataggggaaa tgtcaccacggatgcaggcgaaattactgcgtttccttaatgatggca ctttccgtcgggttggcgaagaccatgaggtgcatgtcgatgtgcggg tgatttgcgctacgcagaagaatctggtcgaactggtgcaaaaaggc atgttccgtgaagatctctattatcgtctgaacgtgttgacgctcaatctg ccgccgctacgtgactgtccgcaggacatcatgccgttaactgagct gttcgtcgcccgctttgccgacgagcagggcgtgccgcgtccgaaa ctggccgctgacctgaatactgtacttacgcgttatgcgtggccggga aatgtgcggcagttaaagaacgctatctatcgcgcactgacacaact ggacggttatgagctgcgtccacaggatattttgttgccggattatgac gccgcaacggtagccgtgggcgaagatgcgatggaaggttcgctg gacgaaatcaccagccgttttgaacgctcggtattaacccagctttatc gcaattatcccagcacgcgcaaactggcaaaacgtctcggcgtttca cataccgcgattgccaataagttgcgggaatatggtctgagtcagaa gaagaacgaagagtaa 17 tyrR_E274Q_D103S ATGcgtctggaagtcttttgtgaagaccgactcggtctgacccgcga attactcgatctactcgtgctaagaggcattgatttacgcggtattgaga ttgatcccattgggcgaatctacctcaattttgctgaactggagtttgag agtttcagcagtctgatggccgaaatacgccgtattgcgggtgttacc gatgtgcgtactgtcccgtggatgccttccgaacgtgagcatctggcgt tgagcgcgttactggaggcgttgcctgaacctgtgctctctgtcgatat gaaaagcaaagtgtccatggcgaacccggcgagctgtcagctttttg ggcaaaaattggatcgcctgcgcaaccataccgccgcacaattgatt aacggctttaattttttacgttggctggaaagcgaaccgcaagattcgc ataacgagcatgtcgttattaatgggcagaatttcctgatggagattac gcctgtttatcttcaggatgaaaatgatcaacacgtcctgaccggtgc ggtggtgatgttgcgatcaacgattcgtatgggccgccagttgcaaaa tgtcgccgcccaggacgtcagcgccttcagtcaaattgtcgccgtca gcccgaaaatgaagcatgttgtcgaacaggcgcagaaactggcga tgctaagcgcgccgctgctgattacgggtgacacaggtacaggtaa agatctctttgcctacgcctgccatcaggcaagccccagagcgggc aaaccttacctggcgctgaactgtgcgtctataccggaagatgcggtc gagagtcaactgtttggtcatgctccggaagggaagaaaggattcttt gagcaggcgaacggtggttcggtgctgttggatgaaataggggaaa tgtcaccacggatgcaggcgaaattactgcgtttccttaatgatggca ctttccgtcgggttggcgaagaccatgaggtgcatgtcgatgtgcggg tgatttgcgctacgcagaagaatctggtcgaactggtgcaaaaaggc atgttccgtgaagatctctattatcgtctgaacgtgttgacgctcaatctg ccgccgctacgtgactgtccgcaggacatcatgccgttaactgagct gttcgtcgcccgctttgccgacgagcagggcgtgccgcgtccgaaa ctggccgctgacctgaatactgtacttacgcgttatgcgtggccggga aatgtgcggcagttaaagaacgctatctatcgcgcactgacacaact ggacggttatgagctgcgtccacaggatattttgttgccggattatgac gccgcaacggtagccgtgggcgaagatgcgatggaaggttcgctg gacgaaatcaccagccgttttgaacgctcggtattaacccagctttatc gcaattatcccagcacgcgcaaactggcaaaacgtctcggcgtttca cataccgcgattgccaataagttgcgggaatatggtctgagtcagaa gaagaacgaagagtaa 18 tyrR_E274Q_C7V ATGcgtctggaagtctttgttgaagaccgactcggtctgacccgcga attactcgatctactcgtgctaagaggcattgatttacgcggtattgaga ttgatcccattgggcgaatctacctcaattttgctgaactggagtttgag agtttcagcagtctgatggccgaaatacgccgtattgcgggtgttacc gatgtgcgtactgtcccgtggatgccttccgaacgtgagcatctggcgt tgagcgcgttactggaggcgttgcctgaacctgtgctctctgtcgatat gaaaagcaaagtggatatggcgaacccggcgagctgtcagctttttg ggcaaaaattggatcgcctgcgcaaccataccgccgcacaattgatt aacggctttaattttttacgttggctggaaagcgaaccgcaagattcgc ataacgagcatgtcgttattaatgggcagaatttcctgatggagattac gcctgtttatcttcaggatgaaaatgatcaacacgtcctgaccggtgc ggtggtgatgttgcgatcaacgattcgtatgggccgccagttgcaaaa tgtcgccgcccaggacgtcagcgccttcagtcaaattgtcgccgtca gcccgaaaatgaagcatgttgtcgaacaggcgcagaaactggcga tgctaagcgcgccgctgctgattacgggtgacacaggtacaggtaa agatctctttgcctacgcctgccatcaggcaagccccagagcgggc aaaccttacctggcgctgaactgtgcgtctataccggaagatgcggtc gagagtcaactgtttggtcatgctccggaagggaagaaaggattcttt gagcaggcgaacggtggttcggtgctgttggatgaaataggggaaa tgtcaccacggatgcaggcgaaattactgcgtttccttaatgatggca ctttccgtcgggttggcgaagaccatgaggtgcatgtcgatgtgcggg tgatttgcgctacgcagaagaatctggtcgaactggtgcaaaaaggc atgttccgtgaagatctctattatcgtctgaacgtgttgacgctcaatctg ccgccgctacgtgactgtccgcaggacatcatgccgttaactgagct gttcgtcgcccgctttgccgacgagcagggcgtgccgcgtccgaaa ctggccgctgacctgaatactgtacttacgcgttatgcgtggccggga aatgtgcggcagttaaagaacgctatctatcgcgcactgacacaact ggacggttatgagctgcgtccacaggatattttgttgccggattatgac gccgcaacggtagccgtgggcgaagatgcgatggaaggttcgctg gacgaaatcaccagccgttttgaacgctcggtattaacccagctttatc gcaattatcccagcacgcgcaaactggcaaaacgtctcggcgtttca cataccgcgattgccaataagttgcgggaatatggtctgagtcagaa gaagaacgaagagtaa 19 tyrR_R10F ATGcgtctggaagtcttttgtgaagactttctcggtctgacccgcgaat tactcgatctactcgtgctaagaggcattgatttacgcggtattgagatt gatcccattgggcgaatctacctcaattttgctgaactggagtttgaga gtttcagcagtctgatggccgaaatacgccgtattgcgggtgttaccg atgtgcgtactgtcccgtggatgccttccgaacgtgagcatctggcgtt gagcgcgttactggaggcgttgcctgaacctgtgctctctgtcgatatg aaaagcaaagtggatatggcgaacccggcgagctgtcagctttttgg gcaaaaattggatcgcctgcgcaaccataccgccgcacaattgatta acggctttaattttttacgttggctggaaagcgaaccgcaagattcgca taacgagcatgtcgttattaatgggcagaatttcctgatggagattacg cctgtttatcttcaggatgaaaatgatcaacacgtcctgaccggtgcg gtggtgatgttgcgatcaacgattcgtatgggccgccagttgcaaaat gtcgccgcccaggacgtcagcgccttcagtcaaattgtcgccgtcag cccgaaaatgaagcatgttgtcgaacaggcgcagaaactggcgat gctaagcgcgccgctgctgattacgggtgacacaggtacaggtaaa gatctctttgcctacgcctgccatcaggcaagccccagagcgggca aaccttacctggcgctgaactgtgcgtctataccggaagatgcggtcg agagtgaactgtttggtcatgctccggaagggaagaaaggattctttg agcaggcgaacggtggttcggtgctgttggatgaaataggggaaat gtcaccacggatgcaggcgaaattactgcgtttccttaatgatggcac tttccgtcgggttggcgaagaccatgaggtgcatgtcgatgtgcgggt gatttgcgctacgcagaagaatctggtcgaactggtgcaaaaaggc atgttccgtgaagatctctattatcgtctgaacgtgttgacgctcaatctg ccgccgctacgtgactgtccgcaggacatcatgccgttaactgagct gttcgtcgcccgctttgccgacgagcagggcgtgccgcgtccgaaa ctggccgctgacctgaatactgtacttacgcgttatgcgtggccggga aatgtgcggcagttaaagaacgctatctatcgcgcactgacacaact ggacggttatgagctgcgtccacaggatattttgttgccggattatgac gccgcaacggtagccgtgggcgaagatgcgatggaaggttcgctg gacgaaatcaccagccgttttgaacgctcggtattaacccagctttatc gcaattatcccagcacgcgcaaactggcaaaacgtctcggcgtttca cataccgcgattgccaataagttgcgggaatatggtctgagtcagaa gaagaacgaagagtaa 20 tyrR_L11G ATGcgtctggaagtcttttgtgaagaccgagggggtctgacccgcg aattactcgatctactcgtgctaagaggcattgatttacgcggtattgag attgatcccattgggcgaatctacctcaattttgctgaactggagtttga gagtttcagcagtctgatggccgaaatacgccgtattgcgggtgttac cgatgtgcgtactgtcccgtggatgccttccgaacgtgagcatctggc gttgagcgcgttactggaggcgttgcctgaacctgtgctctctgtcgat atgaaaagcaaagtggatatggcgaacccggcgagctgtcagctttt tgggcaaaaattggatcgcctgcgcaaccataccgccgcacaattg attaacggctttaattttttacgttggctggaaagcgaaccgcaagattc gcataacgagcatgtcgttattaatgggcagaatttcctgatggagatt acgcctgtttatcttcaggatgaaaatgatcaacacgtcctgaccggt gcggtggtgatgttgcgatcaacgattcgtatgggccgccagttgcaa aatgtcgccgcccaggacgtcagcgccttcagtcaaattgtcgccgt cagcccgaaaatgaagcatgttgtcgaacaggcgcagaaactggc gatgctaagcgcgccgctgctgattacgggtgacacaggtacaggt aaagatctctttgcctacgcctgccatcaggcaagccccagagcgg gcaaaccttacctggcgctgaactgtgcgtctataccggaagatgcg gtcgagagtgaactgtttggtcatgctccggaagggaagaaaggatt ctttgagcaggcgaacggtggttcggtgctgttggatgaaatagggg aaatgtcaccacggatgcaggcgaaattactgcgtttccttaatgatg gcactttccgtcgggttggcgaagaccatgaggtgcatgtcgatgtgc gggtgatttgcgctacgcagaagaatctggtcgaactggtgcaaaaa ggcatgttccgtgaagatctctattatcgtctgaacgtgttgacgctcaa tctgccgccgctacgtgactgtccgcaggacatcatgccgttaactga gctgttcgtcgcccgctttgccgacgagcagggcgtgccgcgtccga aactggccgctgacctgaatactgtacttacgcgttatgcgtggccgg gaaatgtgcggcagttaaagaacgctatctatcgcgcactgacaca actggacggttatgagctgcgtccacaggatattttgttgccggattatg acgccgcaacggtagccgtgggcgaagatgcgatggaaggttcgc tggacgaaatcaccagccgttttgaacgctcggtattaacccagcttta tcgcaattatcccagcacgcgcaaactggcaaaacgtctcggcgttt cacataccgcgattgccaataagttgcgggaatatggtctgagtcag aagaagaacgaagagtaa 21 tyrR_G12T ATGcgtctggaagtcttttgtgaagaccgactcacgctgacccgcg aattactcgatctactcgtgctaagaggcattgatttacgcggtattgag attgatcccattgggcgaatctacctcaattttgctgaactggagtttga gagtttcagcagtctgatggccgaaatacgccgtattgcgggtgttac cgatgtgcgtactgtcccgtggatgccttccgaacgtgagcatctggc gttgagcgcgttactggaggcgttgcctgaacctgtgctctctgtcgat atgaaaagcaaagtggatatggcgaacccggcgagctgtcagctttt tgggcaaaaattggatcgcctgcgcaaccataccgccgcacaattg attaacggctttaattttttacgttggctggaaagcgaaccgcaagattc gcataacgagcatgtcgttattaatgggcagaatttcctgatggagatt acgcctgtttatcttcaggatgaaaatgatcaacacgtcctgaccggt gcggtggtgatgttgcgatcaacgattcgtatgggccgccagttgcaa aatgtcgccgcccaggacgtcagcgccttcagtcaaattgtcgccgt cagcccgaaaatgaagcatgttgtcgaacaggcgcagaaactggc gatgctaagcgcgccgctgctgattacgggtgacacaggtacaggt aaagatctctttgcctacgcctgccatcaggcaagccccagagcgg gcaaaccttacctggcgctgaactgtgcgtctataccggaagatgcg gtcgagagtgaactgtttggtcatgctccggaagggaagaaaggatt ctttgagcaggcgaacggtggttcggtgctgttggatgaaatagggg aaatgtcaccacggatgcaggcgaaattactgcgtttccttaatgatg gcactttccgtcgggttggcgaagaccatgaggtgcatgtcgatgtgc gggtgatttgcgctacgcagaagaatctggtcgaactggtgcaaaaa ggcatgttccgtgaagatctctattatcgtctgaacgtgttgacgctcaa tctgccgccgctacgtgactgtccgcaggacatcatgccgttaactga gctgttcgtcgcccgctttgccgacgagcagggcgtgccgcgtccga aactggccgctgacctgaatactgtacttacgcgttatgcgtggccgg gaaatgtgcggcagttaaagaacgctatctatcgcgcactgacaca actggacggttatgagctgcgtccacaggatattttgttgccggattatg acgccgcaacggtagccgtgggcgaagatgcgatggaaggttcgc tggacgaaatcaccagccgttttgaacgctcggtattaacccagcttta tcgcaattatcccagcacgcgcaaactggcaaaacgtctcggcgttt cacataccgcgattgccaataagttgcgggaatatggtctgagtcag aagaagaacgaagagtaa 22 tyrR_L13G ATGcgtctggaagtcttttgtgaagaccgactcggtggaacccgcg aattactcgatctactcgtgctaagaggcattgatttacgcggtattgag attgatcccattgggcgaatctacctcaattttgctgaactggagtttga gagtttcagcagtctgatggccgaaatacgccgtattgcgggtgttac cgatgtgcgtactgtcccgtggatgccttccgaacgtgagcatctggc gttgagcgcgttactggaggcgttgcctgaacctgtgctctctgtcgat atgaaaagcaaagtggatatggcgaacccggcgagctgtcagctttt tgggcaaaaattggatcgcctgcgcaaccataccgccgcacaattg attaacggctttaattttttacgttggctggaaagcgaaccgcaagattc gcataacgagcatgtcgttattaatgggcagaatttcctgatggagatt acgcctgtttatcttcaggatgaaaatgatcaacacgtcctgaccggt gcggtggtgatgttgcgatcaacgattcgtatgggccgccagttgcaa aatgtcgccgcccaggacgtcagcgccttcagtcaaattgtcgccgt cagcccgaaaatgaagcatgttgtcgaacaggcgcagaaactggc gatgctaagcgcgccgctgctgattacgggtgacacaggtacaggt aaagatctctttgcctacgcctgccatcaggcaagccccagagcgg gcaaaccttacctggcgctgaactgtgcgtctataccggaagatgcg gtcgagagtgaactgtttggtcatgctccggaagggaagaaaggatt ctttgagcaggcgaacggtggttcggtgctgttggatgaaatagggg aaatgtcaccacggatgcaggcgaaattactgcgtttccttaatgatg gcactttccgtcgggttggcgaagaccatgaggtgcatgtcgatgtgc gggtgatttgcgctacgcagaagaatctggtcgaactggtgcaaaaa ggcatgttccgtgaagatctctattatcgtctgaacgtgttgacgctcaa tctgccgccgctacgtgactgtccgcaggacatcatgccgttaactga gctgttcgtcgcccgctttgccgacgagcagggcgtgccgcgtccga aactggccgctgacctgaatactgtacttacgcgttatgcgtggccgg gaaatgtgcggcagttaaagaacgctatctatcgcgcactgacaca actggacggttatgagctgcgtccacaggatattttgttgccggattatg acgccgcaacggtagccgtgggcgaagatgcgatggaaggttcgc tggacgaaatcaccagccgttttgaacgctcggtattaacccagcttta tcgcaattatcccagcacgcgcaaactggcaaaacgtctcggcgttt cacataccgcgattgccaataagttgcgggaatatggtctgagtcag aagaagaacgaagagtaa 23 tyrR_T14K ATGcgtctggaagtcttttgtgaagaccgactcggtctgaagcgcga attactcgatctactcgtgctaagaggcattgatttacgcggtattgaga ttgatcccattgggcgaatctacctcaattttgctgaactggagtttgag agtttcagcagtctgatggccgaaatacgccgtattgcgggtgttacc gatgtgcgtactgtcccgtggatgccttccgaacgtgagcatctggcgt tgagcgcgttactggaggcgttgcctgaacctgtgctctctgtcgatat gaaaagcaaagtggatatggcgaacccggcgagctgtcagctttttg ggcaaaaattggatcgcctgcgcaaccataccgccgcacaattgatt aacggctttaattttttacgttggctggaaagcgaaccgcaagattcgc ataacgagcatgtcgttattaatgggcagaatttcctgatggagattac gcctgtttatcttcaggatgaaaatgatcaacacgtcctgaccggtgc ggtggtgatgttgcgatcaacgattcgtatgggccgccagttgcaaaa tgtcgccgcccaggacgtcagcgccttcagtcaaattgtcgccgtca gcccgaaaatgaagcatgttgtcgaacaggcgcagaaactggcga tgctaagcgcgccgctgctgattacgggtgacacaggtacaggtaa agatctctttgcctacgcctgccatcaggcaagccccagagcgggc aaaccttacctggcgctgaactgtgcgtctataccggaagatgcggtc gagagtgaactgtttggtcatgctccggaagggaagaaaggattcttt gagcaggcgaacggtggttcggtgctgttggatgaaataggggaaa tgtcaccacggatgcaggcgaaattactgcgtttccttaatgatggca ctttccgtcgggttggcgaagaccatgaggtgcatgtcgatgtgcggg tgatttgcgctacgcagaagaatctggtcgaactggtgcaaaaaggc atgttccgtgaagatctctattatcgtctgaacgtgttgacgctcaatctg ccgccgctacgtgactgtccgcaggacatcatgccgttaactgagct gttcgtcgcccgctttgccgacgagcagggcgtgccgcgtccgaaa ctggccgctgacctgaatactgtacttacgcgttatgcgtggccggga aatgtgcggcagttaaagaacgctatctatcgcgcactgacacaact ggacggttatgagctgcgtccacaggatattttgttgccggattatgac gccgcaacggtagccgtgggcgaagatgcgatggaaggttcgctg gacgaaatcaccagccgttttgaacgctcggtattaacccagctttatc gcaattatcccagcacgcgcaaactggcaaaacgtctcggcgtttca cataccgcgattgccaataagttgcgggaatatggtctgagtcagaa gaagaacgaagagtaa TynA-FeaR Sensor 24 PtynA (used in tcggtgaatgaagggcaacggcgaatagttgcccttttatttcactaag pHJY028) ttttgtgacgttgtcacattatgcatgatgtgtacatctattttcagggcatc cactgtatgaaaagctggcacacctgccaaacaacctggcaggtgc aggcaatcccctttgcatcagtactgataatgtgaacctgactaaacc gcccacagagcgcggttgctaacaagaacacaacagaaagagg ggttaata 25 PtynA (used in tatgaaaagctggcacacctgccaaacaacctggcaggtgcaggc pCX008) aatcccctttgcatcagtactgataatgtgaacctgactaaaccgccc acagagcgcggttgctaacaagaacacaacagaaagaggggtta ata 26 gfp RBS library for AgaacacaacaRRRRRRRRRgttaataATG PtynA-gfp reporter 27 Optimal RBS for agaacacaacagaaagagggttaataATG PtynA-gfp reporter (pHJY028) 28 bla RBS library aactaagacgWtctRggBgtcaWHaaATG 29 RBS-bla aactaagacgttctaggcgtcatcaaATGagtattcaacatttccgtg tcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccaga aacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacga gtgggttacatcgaactggatctcaacagcggtaagatccttgagagt tttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgct atgtggcgcggtattatcccgtattgacgccgggcaagagcaactcg gtcgccgcatacactattctcagaatgacttggttgagtactcaccagt cacagaaaagcatcttacggatggcatgacagtaagagaattatgc agtgctgccataaccatgagtgataacactgcggccaacttacttctg acaacgatcggaggaccgaaggagctaaccgcttttttgcacaaca tgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatg aagccataccaaacgacgagcgtgacaccacgatgcctgtagcaa tggcaacaacgttgcgcaaactattaactggcgaactacttactctag cttcccggcaacaattaatagactggatggaggcggataaagttgca ggaccacttctgcgctcggcccttccggctggctggtttattgctgataa atctggagccggtgagcgtgggtctcgcggtatcattgcagcactgg ggccagatggtaagccctcccgtatcgtagttatctacacgacgggg agtcaggcaactatggatgaacgaaatagacagatcgctgagata ggtgcctcactgattaagcattggtaa 30 sacB RBS library caaaatccWcaaDggtRgttgBBgATG 31 RBS-sacB caaaatcctcaaaggtggttgcggATGaacatcaaaaagtttgca aaacaagcaacagtattaacctttactaccgcactgctggcaggag gcgcaactcaagcgtttgcgaaagaaacgaaccaaaagccatata aggaaacatacggcatttcccatattacacgccatgatatgctgcaa atccctgaacagcaaaaaaatgaaaaatatcaagttcctgaattcga ttcgtccacaattaaaaatatctcttctgcaaaaggcctggacgtttgg gacagctggccattacaaaacgctgacggcactgtcgcaaactatc acggctaccacatcgtctttgcattagccggagatcctaaaaatgcgg atgacacatcgatttacatgttctatcaaaaagtcggcgaaacttctatt gacagctggaaaaacgctggccgcgtctttaaagacagcgacaaa ttcgatgcaaatgattctatcctaaaagaccaaacacaagaatggtc aggttcagccacatttacatctgacggaaaaatccgtttattctacact gatttctccggtaaacattacggcaaacaaacactgacaactgcaca agttaacgtatcagcatcagacagctctttgaacatcaacggtgtaga ggattataaatcaatctttgacggtgacggaaaaacgtatcaaaatgt acagcagttcatcgatgaaggcaactacagctcaggcgacaaccat acgctgagagatcctcactacgtagaagataaaggccacaaatact tagtatttgaagcaaacactggaactgaagatggctaccaaggcga agaatctttatttaacaaagcatactatggcaaaagcacatcattcttc cgtcaagaaagtcaaaaacttctgcaaagcgataaaaaacgcacg gctgagttagcaaacggcgctctcggtatgattgagctaaacgatgat tacacactgaaaaaagtgatgaaaccgctgattgcatctaacacagt aacagatgaaattgaacgcgcgaacgtctttaaaatgaacggcaaa tggtatctgttcactgactcccgcggatcaaaaatgacgattgacggc attacgtctaacgatatttacatgcttggttatgtttctaattctttaactggc ccatacaagccgctgaacaaaactggccttgtgttaaaaatggatctt gatcctaacgatgtaacctttacttactcacacttcgctgtacctcaagc gaaaggaaacaatgtcgtgattacaagctatatgacaaacagagg attctacgcagacaaacaatcaacgtttgcgccaagcttcctgctgaa catcaaaggcaagaaaacatctgttgtcaaagacagcatccttgaa caaggacaattaacagttaacaaataa 32 [Pcon-RBS-]tynA [Ttgacagctagctcagtcctaggagttgagctagccttgcccacag agcgcggttgctaacaagaacacaacatctgacgaggttaata]AT Gggaagcccctctctgtattctgcccgtaaaacaaccctggcgttgg cagtcgccttaagtttcgcctggcaagcgccggtatttgcccacggtg gtgaagcgcatatggtgccaatggataaaacgcttaaagaatttggt gccgatgtgcagtgggacgactacgcccagctctttaccctgattaaa gatggcgcgtacgtgaaagtgaagcctggtgcgcaaacagcaattg ttaatggtcagcctctggcactgcaagtaccggtagtgatgaaagac aataaagcctgggtttctgacacctttattaacgatgttttccagtccgg gctggatcaaacctttcaggtagaaaagcgccctcacccacttaatg cgctaactgcggacgaaattaaacaggccgttgaaattgttaaagctt ccgcggacttcaaacccaatacccgttttactgagatctccctgctacc gccagataaagaagctgtctgggcgtttgcgctggaaaacaaaccg gttgaccagccgcgcaaagccgacgtcattatgctcgacggcaaac atatcatcgaagcggtggtggatctgcaaaacaacaaactgctctcc tggcaacccattaaagacgcccacggtatggtgttgctggatgatttc gccagtgtgcagaacattattaacaacagtgaagaatttgccgctgc cgtgaagaaacgcggtattactgatgcgaaaaaagtgattaccacg ccgctgaccgtaggttatttcgatggtaaagatggcctgaaacaagat gcccggttgctcaaagtcatcagctatcttgatgtcggtgatggcaact actgggcacatcccatcgaaaacctggtggcggtcgttgatttagaa cagaaaaaaatcgttaagattgaagaaggtccggtagttccggtgcc aatgaccgcacgcccatttgatggccgtgaccgcgttgctccggcag ttaagcctatgcaaatcattgagcctgaaggtaaaaattacaccatta ctggcgatatgattcactggcggaactgggattttcacctcagcatga actctcgcgtcgggccgatgatctccaccgtgacttataacgacaatg gcaccaaacgcaaagtcatgtacgaaggttctctcggcggcatgatt gtgccttacggtgatcctgatattggctggtactttaaagcgtatctgga ctctggtgactacggtatgggcacgctaacctcaccaattgctcgtggt aaagatgccccgtctaacgcagtgctccttaatgaaaccatcgccga ctacactggcgtgccgatggagatccctcgcgctatcgcggtatttga acgttatgccgggccggagtataagcatcaggaaatgggccagcc caacgtcagtaccgaacgccgggagttagtggtgcgctggatcagt acagtgggtaactatgactacatttttgactggatcttccatgaaaacg gcactattggcatcgatgccggtgctacgggcatcgaagcggtgaa aggtgttaaagcgaaaaccatgcacgatgagacggcgaaagatg acacgcgctacggcacgcttatcgatcacaatatcgtgggtactaca caccaacatatttataatttccgcctcgatctggatgtagatggcgaga ataacagcctggtggcgatggacccagtggtaaaaccgaatactgc cggtggcccacgcaccagtaccatgcaagttaatcagtacaacatc ggcaatgaacaggatgccgcacagaaatttgatccgggcacgattc gtctgttgagtaacccgaacaaagagaaccgcatgggcaatccggt ttcctatcaaattattccttatgcaggtggtactcacccggtagcaaaa ggtgcccagttcgcgccggacgagtggatctatcatcgtttaagcttta tggacaagcagctctgggtaacgcgttatcatcctggcgagcgtttcc cggaaggcaaatatccgaaccgttctactcatgacaccggtcttgga caatacagtaaggataacgagtcgctggacaacaccgacgccgtt gtctggatgaccaccggcaccacacatgtggcccgcgccgaagag tggccgattatgccgaccgaatgggtacatactctgctgaaaccatg gaacttctttgacgaaacgccaacgctaggggcgctgaagaaagat aagtga 33 tynA_G494S ATGggaagcccctctctgtattctgcccgtaaaacaaccctggcgtt ggcagtcgccttaagtttcgcctggcaagcgccggtatttgcccacgg tggtgaagcgcatatggtgccaatggataaaacgcttaaagaatttg gtgccgatgtgcagtgggacgactacgcccagctctttaccctgatta aagatggcgcgtacgtgaaagtgaagcctggtgcgcaaacagcaa ttgttaatggtcagcctctggcactgcaagtaccggtagtgatgaaag acaataaagcctgggtttctgacacctttattaacgatgttttccagtcc gggctggatcaaacctttcaggtagaaaagcgccctcacccacttaa tgcgctaactgcggacgaaattaaacaggccgttgaaattgttaaag cttccgcggacttcaaacccaatacccgttttactgagatctccctgcta ccgccagataaagaagctgtctgggcgtttgcgctggaaaacaaac cggttgaccagccgcgcaaagccgacgtcattatgctcgacggcaa acatatcatcgaagcggtggtggatctgcaaaacaacaaactgctct cctggcaacccattaaagacgcccacggtatggtgttgctggatgatt tcgccagtgtgcagaacattattaacaacagtgaagaatttgccgctg ccgtgaagaaacgcggtattactgatgcgaaaaaagtgattaccac gccgctgaccgtaggttatttcgatggtaaagatggcctgaaacaag atgcccggttgctcaaagtcatcagctatcttgatgtcggtgatggcaa ctactgggcacatcccatcgaaaacctggtggcggtcgttgatttaga acagaaaaaaatcgttaagattgaagaaggtccggtagttccggtg ccaatgaccgcacgcccatttgatggccgtgaccgcgttgctccggc agttaagcctatgcaaatcattgagcctgaaggtaaaaattacaccat tactggcgatatgattcactggcggaactgggattttcacctcagcatg aactctcgcgtcgggccgatgatctccaccgtgacttataacgacaat ggcaccaaacgcaaagtcatgtacgaaggttctctcggcggcatga ttgtgccttacggtgatcctgatattggctggtactttaaagcgtatctgg actctggtgactacggtatgggcacgctaacctcaccaattgctcgtg gtaaagatgccccgtctaacgcagtgctccttaatgaaaccatcgcc gactacactggcgtgccgatggagatccctcgcgctatcgcggtattt gaacgttatgccgggccggagtataagcatcaggaaatgggccag cccaacgtcagtaccgaacgccgggagttagtggtgcgctggatca gtacagtgtccaactatgactacatttttgactggatcttccatgaaaac ggcactattggcatcgatgccggtgctacgggcatcgaagcggtga aaggtgttaaagcgaaaaccatgcacgatgagacggcgaaagat gacacgcgctacggcacgcttatcgatcacaatatcgtgggtactac acaccaacatatttataatttccgcctcgatctggatgtagatggcgag aataacagcctggtggcgatggacccagtggtaaaaccgaatactg ccggtggcccacgcaccagtaccatgcaagttaatcagtacaacat cggcaatgaacaggatgccgcacagaaatttgatccgggcacgatt cgtctgttgagtaacccgaacaaagagaaccgcatgggcaatccg gtttcctatcaaattattccttatgcaggtggtactcacccggtagcaaa aggtgcccagttcgcgccggacgagtggatctatcatcgtttaagcttt atggacaagcagctctgggtaacgcgttatcatcctggcgagcgtttc ccggaaggcaaatatccgaaccgttctactcatgacaccggtcttgg acaatacagtaaggataacgagtcgctggacaacaccgacgccgt tgtctggatgaccaccggcaccacacatgtggcccgcgccgaaga gtggccgattatgccgaccgaatgggtacatactctgctgaaaccat ggaacttctttgacgaaacgccaacgctaggggcgctgaagaaag ataagtga 34 tynA_G415H ATGggaagcccctctctgtattctgcccgtaaaacaaccctggcgtt ggcagtcgccttaagtttcgcctggcaagcgccggtatttgcccacgg tggtgaagcgcatatggtgccaatggataaaacgcttaaagaatttg gtgccgatgtgcagtgggacgactacgcccagctctttaccctgatta aagatggcgcgtacgtgaaagtgaagcctggtgcgcaaacagcaa ttgttaatggtcagcctctggcactgcaagtaccggtagtgatgaaag acaataaagcctgggtttctgacacctttattaacgatgttttccagtcc gggctggatcaaacctttcaggtagaaaagcgccctcacccacttaa tgcgctaactgcggacgaaattaaacaggccgttgaaattgttaaag cttccgcggacttcaaacccaatacccgttttactgagatctccctgcta ccgccagataaagaagctgtctgggcgtttgcgctggaaaacaaac cggttgaccagccgcgcaaagccgacgtcattatgctcgacggcaa acatatcatcgaagcggtggtggatctgcaaaacaacaaactgctct cctggcaacccattaaagacgcccacggtatggtgttgctggatgatt tcgccagtgtgcagaacattattaacaacagtgaagaatttgccgctg ccgtgaagaaacgcggtattactgatgcgaaaaaagtgattaccac gccgctgaccgtaggttatttcgatggtaaagatggcctgaaacaag atgcccggttgctcaaagtcatcagctatcttgatgtcggtgatggcaa ctactgggcacatcccatcgaaaacctggtggcggtcgttgatttaga acagaaaaaaatcgttaagattgaagaaggtccggtagttccggtg ccaatgaccgcacgcccatttgatggccgtgaccgcgttgctccggc agttaagcctatgcaaatcattgagcctgaaggtaaaaattacaccat tactggcgatatgattcactggcggaactgggattttcacctcagcatg aactctcgcgtcgggccgatgatctccaccgtgacttataacgacaat ggcaccaaacgcaaagtcatgtacgaaggttctctcggcggcatga ttgtgccttacggtgatcctgatattggctggtactttaaagcgtatctgg actctcacgactacggtatgggcacgctaacctcaccaattgctcgtg gtaaagatgccccgtctaacgcagtgctccttaatgaaaccatcgcc gactacactggcgtgccgatggagatccctcgcgctatcgcggtattt gaacgttatgccgggccggagtataagcatcaggaaatgggccag cccaacgtcagtaccgaacgccgggagttagtggtgcgctggatca gtacagtgggtaactatgactacatttttgactggatcttccatgaaaac ggcactattggcatcgatgccggtgctacgggcatcgaagcggtga aaggtgttaaagcgaaaaccatgcacgatgagacggcgaaagat gacacgcgctacggcacgcttatcgatcacaatatcgtgggtactac acaccaacatatttataatttccgcctcgatctggatgtagatggcgag aataacagcctggtggcgatggacccagtggtaaaaccgaatactg ccggtggcccacgcaccagtaccatgcaagttaatcagtacaacat cggcaatgaacaggatgccgcacagaaatttgatccgggcacgatt cgtctgttgagtaacccgaacaaagagaaccgcatgggcaatccg gtttcctatcaaattattccttatgcaggtggtactcacccggtagcaaa aggtgcccagttcgcgccggacgagtggatctatcatcgtttaagcttt atggacaagcagctctgggtaacgcgttatcatcctggcgagcgtttc ccggaaggcaaatatccgaaccgttctactcatgacaccggtcttgg acaatacagtaaggataacgagtcgctggacaacaccgacgccgt tgtctggatgaccaccggcaccacacatgtggcccgcgccgaaga gtggccgattatgccgaccgaatgggtacatactctgctgaaaccat ggaacttctttgacgaaacgccaacgctaggggcgctgaagaaag ataagtga 35 tynA_S414M ATGggaagcccctctctgtattctgcccgtaaaacaaccctggcgtt ggcagtcgccttaagtttcgcctggcaagcgccggtatttgcccacgg tggtgaagcgcatatggtgccaatggataaaacgcttaaagaatttg gtgccgatgtgcagtgggacgactacgcccagctctttaccctgatta aagatggcgcgtacgtgaaagtgaagcctggtgcgcaaacagcaa ttgttaatggtcagcctctggcactgcaagtaccggtagtgatgaaag acaataaagcctgggtttctgacacctttattaacgatgttttccagtcc gggctggatcaaacctttcaggtagaaaagcgccctcacccacttaa tgcgctaactgcggacgaaattaaacaggccgttgaaattgttaaag cttccgcggacttcaaacccaatacccgttttactgagatctccctgcta ccgccagataaagaagctgtctgggcgtttgcgctggaaaacaaac cggttgaccagccgcgcaaagccgacgtcattatgctcgacggcaa acatatcatcgaagcggtggtggatctgcaaaacaacaaactgctct cctggcaacccattaaagacgcccacggtatggtgttgctggatgatt tcgccagtgtgcagaacattattaacaacagtgaagaatttgccgctg ccgtgaagaaacgcggtattactgatgcgaaaaaagtgattaccac gccgctgaccgtaggttatttcgatggtaaagatggcctgaaacaag atgcccggttgctcaaagtcatcagctatcttgatgtcggtgatggcaa ctactgggcacatcccatcgaaaacctggtggcggtcgttgatttaga acagaaaaaaatcgttaagattgaagaaggtccggtagttccggtg ccaatgaccgcacgcccatttgatggccgtgaccgcgttgctccggc agttaagcctatgcaaatcattgagcctgaaggtaaaaattacaccat tactggcgatatgattcactggcggaactgggattttcacctcagcatg aactctcgcgtcgggccgatgatctccaccgtgacttataacgacaat ggcaccaaacgcaaagtcatgtacgaaggttctctcggcggcatga ttgtgccttacggtgatcctgatattggctggtactttaaagcgtatctgg acatgggtgactacggtatgggcacgctaacctcaccaattgctcgtg gtaaagatgccccgtctaacgcagtgctccttaatgaaaccatcgcc gactacactggcgtgccgatggagatccctcgcgctatcgcggtattt gaacgttatgccgggccggagtataagcatcaggaaatgggccag cccaacgtcagtaccgaacgccgggagttagtggtgcgctggatca gtacagtgggtaactatgactacatttttgactggatcttccatgaaaac ggcactattggcatcgatgccggtgctacgggcatcgaagcggtga aaggtgttaaagcgaaaaccatgcacgatgagacggcgaaagat gacacgcgctacggcacgcttatcgatcacaatatcgtgggtactac acaccaacatatttataatttccgcctcgatctggatgtagatggcgag aataacagcctggtggcgatggacccagtggtaaaaccgaatactg ccggtggcccacgcaccagtaccatgcaagttaatcagtacaacat cggcaatgaacaggatgccgcacagaaatttgatccgggcacgatt cgtctgttgagtaacccgaacaaagagaaccgcatgggcaatccg gtttcctatcaaattattccttatgcaggtggtactcacccggtagcaaa aggtgcccagttcgcgccggacgagtggatctatcatcgtttaagcttt atggacaagcagctctgggtaacgcgttatcatcctggcgagcgtttc ccggaaggcaaatatccgaaccgttctactcatgacaccggtcttgg acaatacagtaaggataacgagtcgctggacaacaccgacgccgt tgtctggatgaccaccggcaccacacatgtggcccgcgccgaaga gtggccgattatgccgaccgaatgggtacatactctgctgaaaccat ggaacttctttgacgaaacgccaacgctaggggcgctgaagaaag ataagtga 36 [Pcon-RBS-]feaR [ttgacagctagctcagtcctaggtcttgcgctagcgcaacacaaatg caacaataaaaatacatttcacagagcgaaaacgtgcc]ATGaac cccgcagtggataatgagtttcagcaatggctttcccaaatcaatcag gtatgcggaaattttaccggacgcctgctgactgagcgttacacgggt gtactggacacgcattttgccaaaggactaaagctgagtaccgtgac aaccagcggggtgaatttatcccgcacctggcaggaagtaaaagg cagcgacgatgcctggttttacaccgtttttcagcttagtggtcaggca ataatggagcaggatgagcgtcaggtgcagattggcgctggcgata ttacgttactcgatgcctcacgcccctgttcgctttactggcaggagtctt ctaaacagatttcattacttttgccacgcactctgctggaacaatattttc cccatcaaaaacctatctgcgcagaaagactggacgctgacttacc catggtgcaactcagtcatcgcctgttacaggagagcatgaataatc cggcactttctgaaacagaaagtgaagctgcgctacaggcgatggt gtgtctgctgcgcccggtacttcatcagcgggaatctgttcaacctcgt cgtgaacgtcagtttcaaaaagtggttacgttgatagacgataatattc gcgaagagatattacgcccggagtggatagccggagagacaggta tgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttggtagtc gcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcgattcg ccatgccgcagatgatgaaaaactggcaggcatcggctttcattggg gattttctgaccagagtcatttttcaacggtatttaagcaacgctttggga tgacgccaggcgagtatcgacgtaaattccgctaa 37 feaR_A81T ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt cagacaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttactggcag gagtcttctaaacagatttcattacttttgccacgcactctgctggaaca atattttccccatcaaaaacctatctgcgcagaaagactggacgctga cttacccatggtgcaactcagtcatcgcctgttacaggagagcatgaa taatccggcactttctgaaacagaaagtgaagctgcgctacaggcg atggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaac ctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgataa tattcgcgaagagatattacgcccggagtggatagccggagagaca ggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttggt agtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcga ttcgccatgccgcagatgatgaaaaactggcaggcatcggctttcatt ggggattttctgaccagagtcatttttcaacggtatttaagcaacgctttg ggatgacgccaggcgagtatcgacgtaaattccgctaa 38 feaR_A81L ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt cagttgataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttactggcag gagtcttctaaacagatttcattacttttgccacgcactctgctggaaca atattttccccatcaaaaacctatctgcgcagaaagactggacgctga cttacccatggtgcaactcagtcatcgcctgttacaggagagcatgaa taatccggcactttctgaaacagaaagtgaagctgcgctacaggcg atggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaac ctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgataa tattcgcgaagagatattacgcccggagtggatagccggagagaca ggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttggt agtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcga ttcgccatgccgcagatgatgaaaaactggcaggcatcggctttcatt ggggattttctgaccagagtcatttttcaacggtatttaagcaacgctttg ggatgacgccaggcgagtatcgacgtaaattccgctaa 39 feaR_A81P ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt cagcccataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttactggcag gagtcttctaaacagatttcattacttttgccacgcactctgctggaaca atattttccccatcaaaaacctatctgcgcagaaagactggacgctga cttacccatggtgcaactcagtcatcgcctgttacaggagagcatgaa taatccggcactttctgaaacagaaagtgaagctgcgctacaggcg atggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaac ctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgataa tattcgcgaagagatattacgcccggagtggatagccggagagaca ggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttggt agtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcga ttcgccatgccgcagatgatgaaaaactggcaggcatcggctttcatt ggggattttctgaccagagtcatttttcaacggtatttaagcaacgctttg ggatgacgccaggcgagtatcgacgtaaattccgctaa 40 feaR_A81S ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt cagtccataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttactggcag gagtcttctaaacagatttcattacttttgccacgcactctgctggaaca atattttccccatcaaaaacctatctgcgcagaaagactggacgctga cttacccatggtgcaactcagtcatcgcctgttacaggagagcatgaa taatccggcactttctgaaacagaaagtgaagctgcgctacaggcg atggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaac ctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgataa tattcgcgaagagatattacgcccggagtggatagccggagagaca ggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttggt agtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcga ttcgccatgccgcagatgatgaaaaactggcaggcatcggctttcatt ggggattttctgaccagagtcatttttcaacggtatttaagcaacgctttg ggatgacgccaggcgagtatcgacgtaaattccgctaa 41 feaR_A81V ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggtgataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttactggcag gagtcttctaaacagatttcattacttttgccacgcactctgctggaaca atattttccccatcaaaaacctatctgcgcagaaagactggacgctga cttacccatggtgcaactcagtcatcgcctgttacaggagagcatgaa taatccggcactttctgaaacagaaagtgaagctgcgctacaggcg atggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaac ctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgataa tattcgcgaagagatattacgcccggagtggatagccggagagaca ggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttggt agtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcga ttcgccatgccgcagatgatgaaaaactggcaggcatcggctttcatt ggggattttctgaccagagtcatttttcaacggtatttaagcaacgctttg ggatgacgccaggcgagtatcgacgtaaattccgctaa 42 feaR_A81E ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggagataatggagcaggatgagcgtcaggtgcagattggcgct ggcgatattacgttactcgatgcctcacgcccctgttcgctttactggca ggagtcttctaaacagatttcattacttttgccacgcactctgctggaac aatattttccccatcaaaaacctatctgcgcagaaagactggacgctg acttacccatggtgcaactcagtcatcgcctgttacaggagagcatga ataatccggcactttctgaaacagaaagtgaagctgcgctacaggc gatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaa cctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgata atattcgcgaagagatattacgcccggagtggatagccggagagac aggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttgg tagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcg attcgccatgccgcagatgatgaaaaactggcaggcatcggctttca ttggggattttctgaccagagtcatttttcaacggtatttaagcaacgcttt gggatgacgccaggcgagtatcgacgtaaattccgctaa 43 feaR_A81M ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt cagatgataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttactggcag gagtcttctaaacagatttcattacttttgccacgcactctgctggaaca atattttccccatcaaaaacctatctgcgcagaaagactggacgctga cttacccatggtgcaactcagtcatcgcctgttacaggagagcatgaa taatccggcactttctgaaacagaaagtgaagctgcgctacaggcg atggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaac ctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgataa tattcgcgaagagatattacgcccggagtggatagccggagagaca ggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttggt agtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcga ttcgccatgccgcagatgatgaaaaactggcaggcatcggctttcatt ggggattttctgaccagagtcatttttcaacggtatttaagcaacgctttg ggatgacgccaggcgagtatcgacgtaaattccgctaa 44 feaR_A81G ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt cagggcataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttactggcag gagtcttctaaacagatttcattacttttgccacgcactctgctggaaca atattttccccatcaaaaacctatctgcgcagaaagactggacgctga cttacccatggtgcaactcagtcatcgcctgttacaggagagcatgaa taatccggcactttctgaaacagaaagtgaagctgcgctacaggcg atggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaac ctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgataa tattcgcgaagagatattacgcccggagtggatagccggagagaca ggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttggt agtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcga ttcgccatgccgcagatgatgaaaaactggcaggcatcggctttcatt ggggattttctgaccagagtcatttttcaacggtatttaagcaacgctttg ggatgacgccaggcgagtatcgacgtaaattccgctaa 45 feaR_A81I ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt cagatcataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttactggcag gagtcttctaaacagatttcattacttttgccacgcactctgctggaaca atattttccccatcaaaaacctatctgcgcagaaagactggacgctga cttacccatggtgcaactcagtcatcgcctgttacaggagagcatgaa taatccggcactttctgaaacagaaagtgaagctgcgctacaggcg atggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaac ctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgataa tattcgcgaagagatattacgcccggagtggatagccggagagaca ggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttggt agtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcga ttcgccatgccgcagatgatgaaaaactggcaggcatcggctttcatt ggggattttctgaccagagtcatttttcaacggtatttaagcaacgctttg ggatgacgccaggcgagtatcgacgtaaattccgctaa 46 feaR_A81F ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt cagttcataatggagcaggatgagcgtcaggtgcagattggcgctgg cgatattacgttactcgatgcctcacgcccctgttcgctttactggcagg agtcttctaaacagatttcattacttttgccacgcactctgctggaacaat attttccccatcaaaaacctatctgcgcagaaagactggacgctgact tacccatggtgcaactcagtcatcgcctgttacaggagagcatgaat aatccggcactttctgaaacagaaagtgaagctgcgctacaggcga tggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaacct cgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgataat attcgcgaagagatattacgcccggagtggatagccggagagaca ggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttggt agtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcga ttcgccatgccgcagatgatgaaaaactggcaggcatcggctttcatt ggggattttctgaccagagtcatttttcaacggtatttaagcaacgctttg ggatgacgccaggcgagtatcgacgtaaattccgctaa 47 feaR_A81Y ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt cagtacataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttactggcag gagtcttctaaacagatttcattacttttgccacgcactctgctggaaca atattttccccatcaaaaacctatctgcgcagaaagactggacgctga cttacccatggtgcaactcagtcatcgcctgttacaggagagcatgaa taatccggcactttctgaaacagaaagtgaagctgcgctacaggcg atggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaac ctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgataa tattcgcgaagagatattacgcccggagtggatagccggagagaca ggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttggt agtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcga ttcgccatgccgcagatgatgaaaaactggcaggcatcggctttcatt ggggattttctgaccagagtcatttttcaacggtatttaagcaacgctttg ggatgacgccaggcgagtatcgacgtaaattccgctaa 48 feaR_A81W ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt cagtggataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttactggcag gagtcttctaaacagatttcattacttttgccacgcactctgctggaaca atattttccccatcaaaaacctatctgcgcagaaagactggacgctga cttacccatggtgcaactcagtcatcgcctgttacaggagagcatgaa taatccggcactttctgaaacagaaagtgaagctgcgctacaggcg atggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaac ctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgataa tattcgcgaagagatattacgcccggagtggatagccggagagaca ggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttggt agtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcga ttcgccatgccgcagatgatgaaaaactggcaggcatcggctttcatt ggggattttctgaccagagtcatttttcaacggtatttaagcaacgctttg ggatgacgccaggcgagtatcgacgtaaattccgctaa 49 feaR_A81C ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt cagtgcataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttactggcag gagtcttctaaacagatttcattacttttgccacgcactctgctggaaca atattttccccatcaaaaacctatctgcgcagaaagactggacgctga cttacccatggtgcaactcagtcatcgcctgttacaggagagcatgaa taatccggcactttctgaaacagaaagtgaagctgcgctacaggcg atggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaac ctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgataa tattcgcgaagagatattacgcccggagtggatagccggagagaca ggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttggt agtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcga ttcgccatgccgcagatgatgaaaaactggcaggcatcggctttcatt ggggattttctgaccagagtcatttttcaacggtatttaagcaacgctttg ggatgacgccaggcgagtatcgacgtaaattccgctaa 50 feaR_A81N ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt cagaacataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttactggcag gagtcttctaaacagatttcattacttttgccacgcactctgctggaaca atattttccccatcaaaaacctatctgcgcagaaagactggacgctga cttacccatggtgcaactcagtcatcgcctgttacaggagagcatgaa taatccggcactttctgaaacagaaagtgaagctgcgctacaggcg atggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaac ctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgataa tattcgcgaagagatattacgcccggagtggatagccggagagaca ggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttggt agtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcga ttcgccatgccgcagatgatgaaaaactggcaggcatcggctttcatt ggggattttctgaccagagtcatttttcaacggtatttaagcaacgctttg ggatgacgccaggcgagtatcgacgtaaattccgctaa 51 feaR_A81Q ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt cagcagataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttactggcag gagtcttctaaacagatttcattacttttgccacgcactctgctggaaca atattttccccatcaaaaacctatctgcgcagaaagactggacgctga cttacccatggtgcaactcagtcatcgcctgttacaggagagcatgaa taatccggcactttctgaaacagaaagtgaagctgcgctacaggcg atggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaac ctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgataa tattcgcgaagagatattacgcccggagtggatagccggagagaca ggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttggt agtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcga ttcgccatgccgcagatgatgaaaaactggcaggcatcggctttcatt ggggattttctgaccagagtcatttttcaacggtatttaagcaacgctttg ggatgacgccaggcgagtatcgacgtaaattccgctaa 52 feaR_A81K ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt cagaagataatggagcaggatgagcgtcaggtgcagattggcgct ggcgatattacgttactcgatgcctcacgcccctgttcgctttactggca ggagtcttctaaacagatttcattacttttgccacgcactctgctggaac aatattttccccatcaaaaacctatctgcgcagaaagactggacgctg acttacccatggtgcaactcagtcatcgcctgttacaggagagcatga ataatccggcactttctgaaacagaaagtgaagctgcgctacaggc gatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaa cctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgata atattcgcgaagagatattacgcccggagtggatagccggagagac aggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttgg tagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcg attcgccatgccgcagatgatgaaaaactggcaggcatcggctttca ttggggattttctgaccagagtcatttttcaacggtatttaagcaacgcttt gggatgacgccaggcgagtatcgacgtaaattccgctaa 53 feaR_A81H ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt cagcacataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttactggcag gagtcttctaaacagatttcattacttttgccacgcactctgctggaaca atattttccccatcaaaaacctatctgcgcagaaagactggacgctga cttacccatggtgcaactcagtcatcgcctgttacaggagagcatgaa taatccggcactttctgaaacagaaagtgaagctgcgctacaggcg atggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaac ctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgataa tattcgcgaagagatattacgcccggagtggatagccggagagaca ggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttggt agtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcga ttcgccatgccgcagatgatgaaaaactggcaggcatcggctttcatt ggggattttctgaccagagtcatttttcaacggtatttaagcaacgctttg ggatgacgccaggcgagtatcgacgtaaattccgctaa 54 feaR_A81R ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt cagcgcataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttactggcag gagtcttctaaacagatttcattacttttgccacgcactctgctggaaca atattttccccatcaaaaacctatctgcgcagaaagactggacgctga cttacccatggtgcaactcagtcatcgcctgttacaggagagcatgaa taatccggcactttctgaaacagaaagtgaagctgcgctacaggcg atggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaac ctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgataa tattcgcgaagagatattacgcccggagtggatagccggagagaca ggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttggt agtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcga ttcgccatgccgcagatgatgaaaaactggcaggcatcggctttcatt ggggattttctgaccagagtcatttttcaacggtatttaagcaacgctttg ggatgacgccaggcgagtatcgacgtaaattccgctaa 55 feaR_A81D ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggacataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttactggcag gagtcttctaaacagatttcattacttttgccacgcactctgctggaaca atattttccccatcaaaaacctatctgcgcagaaagactggacgctga cttacccatggtgcaactcagtcatcgcctgttacaggagagcatgaa taatccggcactttctgaaacagaaagtgaagctgcgctacaggcg atggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaac ctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgataa tattcgcgaagagatattacgcccggagtggatagccggagagaca ggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttggt agtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcga ttcgccatgccgcagatgatgaaaaactggcaggcatcggctttcatt ggggattttctgaccagagtcatttttcaacggtatttaagcaacgctttg ggatgacgccaggcgagtatcgacgtaaattccgctaa 56 feaR_M83G ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataGGCgagcaggatgagcgtcaggtgcagattggcg ctggcgatattacgttactcgatgcctcacgcccctgttcgctttactgg caggagtcttctaaacagatttcattacttttgccacgcactctgctgga acaatattttccccatcaaaaacctatctgcgcagaaagactggacg ctgacttacccatggtgcaactcagtcatcgcctgttacaggagagca tgaataatccggcactttctgaaacagaaagtgaagctgcgctacag gcgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttc aacctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacg ataatattcgcgaagagatattacgcccggagtggatagccggaga gacaggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggt ttggtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagat gcgattcgccatgccgcagatgatgaaaaactggcaggcatcggct ttcattggggattttctgaccagagtcatttttcaacggtatttaagcaac gctttgggatgacgccaggcgagtatcgacgtaaattccgctaa 57 feaR_M83A ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataGCGgagcaggatgagcgtcaggtgcagattggcg ctggcgatattacgttactcgatgcctcacgcccctgttcgctttactgg caggagtcttctaaacagatttcattacttttgccacgcactctgctgga acaatattttccccatcaaaaacctatctgcgcagaaagactggacg ctgacttacccatggtgcaactcagtcatcgcctgttacaggagagca tgaataatccggcactttctgaaacagaaagtgaagctgcgctacag gcgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttc aacctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacg ataatattcgcgaagagatattacgcccggagtggatagccggaga gacaggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggt ttggtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagat gcgattcgccatgccgcagatgatgaaaaactggcaggcatcggct ttcattggggattttctgaccagagtcatttttcaacggtatttaagcaac gctttgggatgacgccaggcgagtatcgacgtaaattccgctaa 58 feaR_M83P ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataCCCgagcaggatgagcgtcaggtgcagattggcgc tggcgatattacgttactcgatgcctcacgcccctgttcgctttactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 59 feaR_M83V ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataGTTgagcaggatgagcgtcaggtgcagattggcgc tggcgatattacgttactcgatgcctcacgcccctgttcgctttactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 60 feaR_M83I ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataATCgagcaggatgagcgtcaggtgcagattggcgc tggcgatattacgttactcgatgcctcacgcccctgttcgctttactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 61 feaR_M83L ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataTTGgagcaggatgagcgtcaggtgcagattggcgc tggcgatattacgttactcgatgcctcacgcccctgttcgctttactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 62 feaR_M83F ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataTTCgagcaggatgagcgtcaggtgcagattggcgct ggcgatattacgttactcgatgcctcacgcccctgttcgctttactggca ggagtcttctaaacagatttcattacttttgccacgcactctgctggaac aatattttccccatcaaaaacctatctgcgcagaaagactggacgctg acttacccatggtgcaactcagtcatcgcctgttacaggagagcatga ataatccggcactttctgaaacagaaagtgaagctgcgctacaggc gatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaa cctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgata atattcgcgaagagatattacgcccggagtggatagccggagagac aggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttgg tagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcg attcgccatgccgcagatgatgaaaaactggcaggcatcggctttca ttggggattttctgaccagagtcatttttcaacggtatttaagcaacgcttt gggatgacgccaggcgagtatcgacgtaaattccgctaa 63 feaR_M83Y ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataTACgagcaggatgagcgtcaggtgcagattggcgc tggcgatattacgttactcgatgcctcacgcccctgttcgctttactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 64 feaR_M83W ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataTGGgagcaggatgagcgtcaggtgcagattggcgc tggcgatattacgttactcgatgcctcacgcccctgttcgTGGtactg gcaggagtcttctaaacagatttcattacttttgccacgcactctgctgg aacaatattttccccatcaaaaacctatctgcgcagaaagactggac gctgacttacccatggtgcaactcagtcatcgcctgttacaggagagc atgaataatccggcactttctgaaacagaaagtgaagctgcgctaca ggcgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgtt caacctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagac gataatattcgcgaagagatattacgcccggagtggatagccggag agacaggtatgtcagtacgtagtttgtaccgaatgtttgccgataaag gtttggtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcag atgcgattcgccatgccgcagatgatgaaaaactggcaggcatcgg ctttcattggggattttctgaccagagtcatttttcaacggtatttaagcaa cgctttgggatgacgccaggcgagtatcgacgtaaattccgctaa 65 feaR_M83S ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataTCCgagcaggatgagcgtcaggtgcagattggcgc tggcgatattacgttactcgatgcctcacgcccctgttcgctttactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 66 feaR_M83C ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataTGCgagcaggatgagcgtcaggtgcagattggcgc tggcgatattacgttactcgatgcctcacgcccctgttcgctttactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 67 feaR_M83T ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataACGgagcaggatgagcgtcaggtgcagattggcgc tggcgatattacgttactcgatgcctcacgcccctgttcgctttactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 68 feaR_M83N ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataAACgagcaggatgagcgtcaggtgcagattggcgc tggcgatattacgttactcgatgcctcacgcccctgttcgctttactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 69 feaR_M83Q ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataCAAgagcaggatgagcgtcaggtgcagattggcgc tggcgatattacgttactcgatgcctcacgcccctgttcgctttactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 70 feaR_M83H ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataCACgagcaggatgagcgtcaggtgcagattggcgc tggcgatattacgttactcgatgcctcacgcccctgttcgctttactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 71 feaR_M83K ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataAAGgagcaggatgagcgtcaggtgcagattggcgc tggcgatattacgttactcgatgcctcacgcccctgttcgctttactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 72 feaR_M83R ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataCGCgagcaggatgagcgtcaggtgcagattggcgc tggcgatattacgttactcgatgcctcacgcccctgttcgctttactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 73 feaR_M83D ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataGATgagcaggatgagcgtcaggtgcagattggcgc tggcgatattacgttactcgatgcctcacgcccctgttcgctttactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 74 feaR_M83E ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataGAGgagcaggatgagcgtcaggtgcagattggcgc tggcgatattacgttactcgatgcctcacgcccctgttcgctttactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 75 feaR_L108G ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgGGGtactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 76 feaR_L108A ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgGCGtactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 77 feaR_L108P ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgGCGtactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 78 feaR_L108V ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgGTCtactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 79 feaR_L108M ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgACGtactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 80 feaR_L108I ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgATCtactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 81 feaR_L108F ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgTTTtactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 82 feaR_L108Y ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgTACtactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 83 feaR_L108W ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgTGGtactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 84 feaR_L108S ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgTCCtactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 85 feaR_L108C ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgTGCtactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 86 feaR_L108T ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgACGtactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 87 feaR_L108N ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgAACtactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 88 feaR_L108Q ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgCAGtactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 89 feaR_L108H ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgCACtactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 90 feaR_L108K ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgAAAtactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 91 feaR_L108R ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgCGTtactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 92 feaR_L108D ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgGACtactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 93 feaR_L108E ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgGAGtactggc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 94 feaR_W110G ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttacGGCc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 95 feaR_W110A ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttacGCGc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 96 feaR_W110P ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttacCCGc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 97 feaR_W110V ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttacGTGc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 98 feaR_W110M ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttacATGca ggagtcttctaaacagatttcattacttttgccacgcactctgctggaac aatattttccccatcaaaaacctatctgcgcagaaagactggacgctg acttacccatggtgcaactcagtcatcgcctgttacaggagagcatga ataatccggcactttctgaaacagaaagtgaagctgcgctacaggc gatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaa cctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgata atattcgcgaagagatattacgcccggagtggatagccggagagac aggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttgg tagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcg attcgccatgccgcagatgatgaaaaactggcaggcatcggctttca ttggggattttctgaccagagtcatttttcaacggtatttaagcaacgcttt gggatgacgccaggcgagtatcgacgtaaattccgctaa 99 feaR_W110I ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttacATAca ggagtcttctaaacagatttcattacttttgccacgcactctgctggaac aatattttccccatcaaaaacctatctgcgcagaaagactggacgctg acttacccatggtgcaactcagtcatcgcctgttacaggagagcatga ataatccggcactttctgaaacagaaagtgaagctgcgctacaggc gatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaa cctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgata atattcgcgaagagatattacgcccggagtggatagccggagagac aggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttgg tagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcg attcgccatgccgcagatgatgaaaaactggcaggcatcggctttca ttggggattttctgaccagagtcatttttcaacggtatttaagcaacgcttt gggatgacgccaggcgagtatcgacgtaaattccgctaa 100 feaR_W110L ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttacCTCca ggagtcttctaaacagatttcattacttttgccacgcactctgctggaac aatattttccccatcaaaaacctatctgcgcagaaagactggacgctg acttacccatggtgcaactcagtcatcgcctgttacaggagagcatga ataatccggcactttctgaaacagaaagtgaagctgcgctacaggc gatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaa cctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgata atattcgcgaagagatattacgcccggagtggatagccggagagac aggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttgg tagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcg attcgccatgccgcagatgatgaaaaactggcaggcatcggctttca ttggggattttctgaccagagtcatttttcaacggtatttaagcaacgcttt gggatgacgccaggcgagtatcgacgtaaattccgctaa 101 feaR_W110F ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttacTTCca ggagtcttctaaacagatttcattacttttgccacgcactctgctggaac aatattttccccatcaaaaacctatctgcgcagaaagactggacgctg acttacccatggtgcaactcagtcatcgcctgttacaggagagcatga ataatccggcactttctgaaacagaaagtgaagctgcgctacaggc gatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaa cctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgata atattcgcgaagagatattacgcccggagtggatagccggagagac aggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttgg tagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcg attcgccatgccgcagatgatgaaaaactggcaggcatcggctttca ttggggattttctgaccagagtcatttttcaacggtatttaagcaacgcttt gggatgacgccaggcgagtatcgacgtaaattccgctaa 102 feaR_W110Y ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttacTACca ggagtcttctaaacagatttcattacttttgccacgcactctgctggaac aatattttccccatcaaaaacctatctgcgcagaaagactggacgctg acttacccatggtgcaactcagtcatcgcctgttacaggagagcatga ataatccggcactttctgaaacagaaagtgaagctgcgctacaggc gatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaa cctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgata atattcgcgaagagatattacgcccggagtggatagccggagagac aggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttgg tagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcg attcgccatgccgcagatgatgaaaaactggcaggcatcggctttca ttggggattttctgaccagagtcatttttcaacggtatttaagcaacgcttt gggatgacgccaggcgagtatcgacgtaaattccgctaa 103 feaR_W110S ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttacTCCca ggagtcttctaaacagatttcattacttttgccacgcactctgctggaac aatattttccccatcaaaaacctatctgcgcagaaagactggacgctg acttacccatggtgcaactcagtcatcgcctgttacaggagagcatga ataatccggcactttctgaaacagaaagtgaagctgcgctacaggc gatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaa cctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgata atattcgcgaagagatattacgcccggagtggatagccggagagac aggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttgg tagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcg attcgccatgccgcagatgatgaaaaactggcaggcatcggctttca ttggggattttctgaccagagtcatttttcaacggtatttaagcaacgcttt gggatgacgccaggcgagtatcgacgtaaattccgctaa 104 feaR_W110C ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttacTGTca ggagtcttctaaacagatttcattacttttgccacgcactctgctggaac aatattttccccatcaaaaacctatctgcgcagaaagactggacgctg acttacccatggtgcaactcagtcatcgcctgttacaggagagcatga ataatccggcactttctgaaacagaaagtgaagctgcgctacaggc gatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaa cctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgata atattcgcgaagagatattacgcccggagtggatagccggagagac aggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttgg tagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcg attcgccatgccgcagatgatgaaaaactggcaggcatcggctttca ttggggattttctgaccagagtcatttttcaacggtatttaagcaacgcttt gggatgacgccaggcgagtatcgacgtaaattccgctaa 105 feaR_W110T ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttacACCca ggagtcttctaaacagatttcattacttttgccacgcactctgctggaac aatattttccccatcaaaaacctatctgcgcagaaagactggacgctg acttacccatggtgcaactcagtcatcgcctgttacaggagagcatga ataatccggcactttctgaaacagaaagtgaagctgcgctacaggc gatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaa cctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgata atattcgcgaagagatattacgcccggagtggatagccggagagac aggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttgg tagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcg attcgccatgccgcagatgatgaaaaactggcaggcatcggctttca ttggggattttctgaccagagtcatttttcaacggtatttaagcaacgcttt gggatgacgccaggcgagtatcgacgtaaattccgctaa 106 feaR_W110N ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttacAACca ggagtcttctaaacagatttcattacttttgccacgcactctgctggaac aatattttccccatcaaaaacctatctgcgcagaaagactggacgctg acttacccatggtgcaactcagtcatcgcctgttacaggagagcatga ataatccggcactttctgaaacagaaagtgaagctgcgctacaggc gatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaa cctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgata atattcgcgaagagatattacgcccggagtggatagccggagagac aggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttgg tagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcg attcgccatgccgcagatgatgaaaaactggcaggcatcggctttca ttggggattttctgaccagagtcatttttcaacggtatttaagcaacgcttt gggatgacgccaggcgagtatcgacgtaaattccgctaa 107 feaR_W110Q ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttacCAGc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 108 feaR_W110H ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttacCACca ggagtcttctaaacagatttcattacttttgccacgcactctgctggaac aatattttccccatcaaaaacctatctgcgcagaaagactggacgctg acttacccatggtgcaactcagtcatcgcctgttacaggagagcatga ataatccggcactttctgaaacagaaagtgaagctgcgctacaggc gatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaa cctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgata atattcgcgaagagatattacgcccggagtggatagccggagagac aggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttgg tagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcg attcgccatgccgcagatgatgaaaaactggcaggcatcggctttca ttggggattttctgaccagagtcatttttcaacggtatttaagcaacgcttt gggatgacgccaggcgagtatcgacgtaaattccgctaa 109 feaR_W110K ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttacAAGca ggagtcttctaaacagatttcattacttttgccacgcactctgctggaac aatattttccccatcaaaaacctatctgcgcagaaagactggacgctg acttacccatggtgcaactcagtcatcgcctgttacaggagagcatga ataatccggcactttctgaaacagaaagtgaagctgcgctacaggc gatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaa cctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgata atattcgcgaagagatattacgcccggagtggatagccggagagac aggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttgg tagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcg attcgccatgccgcagatgatgaaaaactggcaggcatcggctttca ttggggattttctgaccagagtcatttttcaacggtatttaagcaacgcttt gggatgacgccaggcgagtatcgacgtaaattccgctaa 110 feaR_W110R ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttacCGCc aggagtcttctaaacagatttcattacttttgccacgcactctgctggaa caatattttccccatcaaaaacctatctgcgcagaaagactggacgct gacttacccatggtgcaactcagtcatcgcctgttacaggagagcatg aataatccggcactttctgaaacagaaagtgaagctgcgctacagg cgatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttca acctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgat aatattcgcgaagagatattacgcccggagtggatagccggagaga caggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttg gtagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgc gattcgccatgccgcagatgatgaaaaactggcaggcatcggctttc attggggattttctgaccagagtcatttttcaacggtatttaagcaacgct ttgggatgacgccaggcgagtatcgacgtaaattccgctaa 111 feaR_W110D ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttacGATca ggagtcttctaaacagatttcattacttttgccacgcactctgctggaac aatattttccccatcaaaaacctatctgcgcagaaagactggacgctg acttacccatggtgcaactcagtcatcgcctgttacaggagagcatga ataatccggcactttctgaaacagaaagtgaagctgcgctacaggc gatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaa cctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgata atattcgcgaagagatattacgcccggagtggatagccggagagac aggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttgg tagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcg attcgccatgccgcagatgatgaaaaactggcaggcatcggctttca ttggggattttctgaccagagtcatttttcaacggtatttaagcaacgcttt gggatgacgccaggcgagtatcgacgtaaattccgctaa 112 feaR_W110E ATGaaccccgcagtggataatgagtttcagcaatggctttcccaaat caatcaggtatgcggaaattttaccggacgcctgctgactgagcgtta cacgggtgtactggacacgcattttgccaaaggactaaagctgagta ccgtgacaaccagcggggtgaatttatcccgcacctggcaggaagt aaaaggcagcgacgatgcctggttttacaccgtttttcagcttagtggt caggcaataatggagcaggatgagcgtcaggtgcagattggcgctg gcgatattacgttactcgatgcctcacgcccctgttcgctttacGAAca ggagtcttctaaacagatttcattacttttgccacgcactctgctggaac aatattttccccatcaaaaacctatctgcgcagaaagactggacgctg acttacccatggtgcaactcagtcatcgcctgttacaggagagcatga ataatccggcactttctgaaacagaaagtgaagctgcgctacaggc gatggtgtgtctgctgcgcccggtacttcatcagcgggaatctgttcaa cctcgtcgtgaacgtcagtttcaaaaagtggttacgttgatagacgata atattcgcgaagagatattacgcccggagtggatagccggagagac aggtatgtcagtacgtagtttgtaccgaatgtttgccgataaaggtttgg tagtcgcgcaatatattcgtaaccgtcgtctcgatttttgtgcagatgcg attcgccatgccgcagatgatgaaaaactggcaggcatcggctttca ttggggattttctgaccagagtcatttttcaacggtatttaagcaacgcttt gggatgacgccaggcgagtatcgacgtaaattccgctaa TrpR sensor 113 trpR (WT) atggcccaacaatcaccctattcagcagcgatggcagaacagcgtc accaggagtggttacgttttgtcgacctgcttaagaatgcctaccaaa acgatctccatttaccgttgttaaacctgatgctgacgccagatgagcg cgaagcgttggggactcgcgtgcgtattgtcgaagagctgttgcgcg gcgaaatgagccagcgtgagttaaaaaatgaactcggcgcaggca tcgcgacgattacgcgtggatctaacagcctgaaagccgcgcccgt cgagctgcgccagtggctggaagaggtgttgctgaaaagcgattga 114 trpR (OD) atggcccaacaatcaccctattcagcagcgatggcagaacagcgtc accaggagtggttacgttttgtcgacctgcttaagaatgcctaccaaa acgatctccatttaccgttgttaaacctgatgctgacgccagatgagcg cgaagcgttggggactcgcgtgcgtattgtcgaagagctgttgcgcg gcgaaatgagccagcgtgagttaagcaatgaactcggcgcatgctg cctgacgattcgccgtggatctaacagcctgaaagccgcgcccgtc gagctgcgccagtggctggaagaggtgttgctgaaaagcgattga 115 trpR (O1) atggcccaacaatcaccctattcagcagcgatggcagaacagcgtc accaggagtggttacgttttgtcgacctgcttaagaatgcctaccaaa acgatctccatttaccgttgttaaacctgatgctgacgccagatgagcg cgaagcgttggggactcgcgtgcgtattgtcgaagagctgttgcgcg gcgaaatgagccagcgtgagttaagcaatgaactcggcgcaaact ggcgcacgattaagcgtggatctaacagcctgaaagccgcgcccgt cgagctgcgccagtggctggaagaggtgttgctgaaaagcgattga 116 trpR with RBS aaccgggggaggcattttgcttcccccgctaacaatggcgacatatt ATGgcccaacaatcaccctattcagcagcgatggcagaacagcg tcaccaggagtggttacgttttgtcgacctgcttaagaatgcctaccaa aacgatctccatttaccgttgttaaacctgatgctgacgccagat gagcgcgaagcgttggggactcgcgtgcgtattgtcgaagagctgtt gcgcggcgaaatgagccagcgtgagttaaaaaatgaactcggcgc aggcatcgcgacgattacgcgtggatctaacagcctgaaagccgcg cccgtcgagctgcgccagtggctggaagaggtgttgctgaa aagcgattga 117 trpR (OD) with aaccgggggaggcattttgcttcccctgctaacaaaggagacattttA RBS TGgcccaacaatcaccctattcagcagcgatggcagaacagcgtc accaggagtggttacgttttgtcgacctgcttaagaatgcctaccaaa acgatctccatttaccgttgttaaacctgatgctgacgccagat gagcgcgaagcgttggggactcgcgtgcgtattgtcgaagagctgtt gcgcggcgaaatgagccagcgtgagttaaaaaatgaactcggcgc aggcatcgcgacgattacgcgtggatctaacagcctgaaagccgcg cccgtcgagctgcgccagtggctggaagaggtgttgctgaa aagcgattga 118 trpR (O1) with aaccgggggaggcattttgcttcccccgctaacaatggcgacatatt RBS ATGgcccaacaatcaccctattcagcagcgatggcagaacagcg tcaccaggagtggttacgttttgtcgacctgcttaagaatgcctaccaa aacgatctccatttaccgttgttaaacctgatgctgacgccagat gagcgcgaagcgttggggactcgcgtgcgtattgtcgaagagctgtt gcgcggcgaaatgagccagcgtgagttaagcaatgaactcggcgc aaactggcgcacgattaagcgtggatctaacagcctgaaagccgc gcccgtcgagctgcgccagtggctggaagaggtgttgctgaa aagcgattga 119 Ptrp gagctgttgacaattaatcatcgaactagttaactagtacgc 120 Ptrp(O.sub.D) gagctgttgacaattaatcatcgagtaagttaacttacacgc 121 Ptrp(O.sub.1) gagctgttgacaattaatcatcgatgtagttaactacaacgc Reference (WT) Amino Acid Sequences 122 trpR (WT) MAQQSPYSAAMAEQRHQEWLRFVDLLKNAYQN DLHLPLLNLMLTPDEREALGTRVRIVEE LLRGEMSQRELKNELGAGIATITRGSNSLKAAPVE LRQWLEEVLLKSD 123 tynA (WT) MGSPSLYSARKTTLALAVALSFAWQAPVFAHGG EAHMVPMDKTLKEFGADVQWDDYAQLF TLIKDGAYVKVKPGAQTAIVNGQPLALQVPVVMK DNKAWVSDTFINDVFQSGLDQTFQVE KRPHPLNALTADEIKQAVEIVKASADFKPNTRFTEI SLLPPDKEAVWAFALENKPVDQPR KADVIMLDGKHIIEAVVDLQNNKLLSWQPIKDAHG MVLLDDFASVQNIINNSEEFAAAVK KRGITDAKKVITTPLTVGYFDGKDGLKQDARLLKV ISYLDVGDGNYWAHPIENLVAVVDL EQKKIVKIEEGPVVPVPMTARPFDGRDRVAPAVK PMQIIEPEGKNYTITGDMIHWRNWDF HLSMNSRVGPMISTVTYNDNGTKRKVMYEGSLG GMIVPYGDPDIGWYFKAYLDSGDYGMG TLTSPIARGKDAPSNAVLLNETIADYTGVPMEIPR AIAVFERYAGPEYKHQEMGQPNVST ERRELVVRWISTVGNYDYIFDWIFHENGTIGIDAG ATGIEAVKGVKAKTMHDETAKDDTR YGTLIDHNIVGTTHQHIYNFRLDLDVDGENNSLVA MDPVVKPNTAGGPRTSTMQVNQYNI GNEQDAAQKFDPGTIRLLSNPNKENRMGNPVSY QIIPYAGGTHPVAKGAQFAPDEWIYHR LSFMDKQLWVTRYHPGERFPEGKYPNRSTHDTG LGQYSKDNESLDNTDAVVWMTTGTTHV ARAEEWPIMPTEWVHTLLKPWNFFDETPTLGALK KDK 124 tyrR (WT) MRLEVFCEDRLGLTRELLDLLVLRGIDLRGIEIDPI GRIYLNFAELEFESFSSLMAEIRR IAGVTDVRTVPWMPSEREHLALSALLEALPEPVL SVDMKSKVDMANPASCQLFGQKLDRL RNHTAAQLINGFNFLRWLESEPQDSHNEHVVING QNFLMEITPVYLQDENDQHVLTGAVV MLRSTIRMGRQLQNVAAQDVSAFSQIVAVSPKMK HVVEQAQKLAMLSAPLLITGDTGTGK DLFAYACHQASPRAGKPYLALNCASIPEDAVESE LFGHAPEGKKGFFEQANGGSVLLDEI GEMSPRMQAKLLRFLNDGTFRRVGEDHEVHVDV RVICATQKNLVELVQKGMFREDLYYRL NVLTLNLPPLRDCPQDIMPLTELFVARFADEQGV PRPKLAADLNTVLTRYAWPGNVRQLK NAIYRALTQLDGYELRPQDILLPDYDAATVAVGED AMEGSLDEITSRFERSVLTQLYRNY PSTRKLAKRLGVSHTAIANKLREYGLSQKKNEE 125 feaR (WT) MNPAVDNEFQQWLSQINQVCGNFTGRLLTERYT GVLETHFAKGLKLSTVTTNGVNLYRTW QEIKGSDDAWFYTVFQLSGQAIMEQDERQVQIGA GDITLLDASRPCSLYWQESSKQISLL LPRTLLEQYFPHQKPVCAERLDADLPMVQLSHRL LQESMNNPALSETESEAALQAMVCLL RPVLHQRESVQPRRERQFQKVVTLIDDNIREEILR PEWIAGETGMSVRSLYRMFADKGLV VAQYIRNRRLDFCADAIRHAADDEKLAGIGFHWG FSDQSHFSTVFKQRFGMTPGEYRRKF R
[0208] All plasmids were assembled in E. coli DH10B using the Gibson Assembly or Golden Gate Assembly methods. EcN was transformed with the purified and sequence-verified plasmids for testing. The EcN strain used in this work lacks its native plasmids (obtained from DSMZ). The tynA, feaR, and tyrR genes and PtynA, PtyrP, PtyrR, Pmtr, PtyrB, ParoF, and ParoP promoters were obtained from E. coli MG1655 genomic DNA. pHJY23 containing the inactivated tynA gene (see e.g.,
[0209] Plasmid DNA was isolated using the PureLink Quick Plasmid Miniprep Kit (Invitrogen, Walthem, Mass., USA), and PCR products were extracted from electrophoresis gels using the Zymoclean Gel DNA Recovery Kit (ZYMO research, Irvine, Calif., USA). Enzymes were purchased from New England Biolabs (Ipswich, Mass., USA). Chemicals were purchased from Sigma-Aldrich (St. Louis, Mo., USA) or Gold Biotechnology (Olivette, Mo., USA). All sequencing was performed by Genewiz (South Plainfield, N.J., USA). Primers were purchased from Integrated DNA Technologies (Coralville, Iowa, USA).
[0210] Aromatic Amino Acid Sensing Assay
[0211] Everything but (Eb) medium was prepared by supplementing M9 minimal medium with 1 mM MgSO4, 100 mM CaCl2), 0.4% w/v glucose, and all non-aromatic amino acids (0.8 mM alanine, 5 mM arginine, 0.4 mM asparagine, 0.4 mM aspartate, 0.1 mM cysteine, 0.6 mM glutamate, 0.6 mM glutamine, 0.8 mM glycine, 0.2 mM histidine, 0.4 mM isoleucine, 0.8 mM leucine, 0.4 mM lysine, 0.2 mM methionine, 0.4 mM proline, 10 mM serine, 0.4 mM threonine, and 0.6 mM valine). Single colonies of EcN containing the relevant sensor plasmids were transferred to 5 mL Eb medium in 14 mL round bottom tubes and incubated overnight at 250 rpm and 37° C. Experimental cultures were prepared by diluting overnight cultures 200× into 0.6 mL fresh Eb medium supplemented with the respective ligands (Phe and Tyr) in 2 mL 96-deep well plates (Eppendorf, Hamburg, Germany). Cultures were grown for 8 h at 37° C. and 250 rpm before sampled for fluorimetry or flow cytometry analysis. All medium was supplemented with the relevant antibiotics for plasmid maintenance (34 mg/ml chloramphenicol and 100 mg/ml spectinomycin).
[0212] Aromatic Amine Sensing Assays
[0213] For fluorescence-based quantification, single colonies were transferred to 5 mL LB medium (VWR, Radnor, Pa., USA) in 14 mL round bottom tubes and incubated overnight at 250 rpm and 37° C. Unless otherwise specified, experimental cultures were prepared by diluting overnight cultures 100× into 0.6 mL M9 minimal medium supplemented with 1 mM MgSO4, 100 mM CaCl2), and 2% w/v glycerol as well as the respective ligands (DA, PEA, Tyra, and Trypta) in 2 mL 96-deep well plates. Cultures were grown for 24 h at 37° C. and 250 rpm. After 5 h and 24 h of incubation, samples were obtained from the cultures for flow cytometry analysis.
[0214] For growth-based library screening, b/a (encoding b-lactamase) and sacB (encoding levansucrase) were incorporated downstream of gfp in the pHJY028 reporter plasmid (see e.g., TABLE 4). For optimization, ribosome binding site libraries were designed for b/a and sacB using the RBS Calculator (see e.g., TABLE 6). The optimization resulted in reporter pCX008. To test sensor variants using the pCX008 reporter, single colonies were transferred to 0.6 mL LB medium in 2 mL 96-deep well plates and incubated overnight at 250 rpm and 37° C. The overnight cultures were diluted 50× into 0.6 mL M9 minimal medium supplemented with 1 mM MgSO4, 100 mM CaCl2, and 2% w/v glycerol in 2 mL 96-well deep well plates. For positive selection, cultures were supplemented with 300 mg/mL carbenicillin. Cultures were then incubated for 2 h at 250 rpm and 37° C. After the 2 h incubation, 2 mL of cells were plated onto M9 agar plates supplemented with 2% glycerol and either 300 mg/mL carbenicillin and 1 mM of the amine of interest (positive selection) or 5% (w/v) sucrose and 1 mM of the non-desired amines (negative selection). Plates were incubated overnight at 37° C.
[0215] Fluorimetry
[0216] 200 mL culture samples were collected and transferred to 96-well assay microplates (clear, flat bottom black, Greiner Bio-One). The fluorescence and culture absorbance (Abs) were measured using a Tecan microplate reader (Infinite M200 Pro) as previously described. The fluorescence of GFP was measured with an excitation at 483 nm and emission at 530 nm. The Abs of the samples was measured at 600 nm. The measured fluorescence was normalized by dividing by the Abs and subtracting the same ratio obtained from non-fluorescent wild-type cells (Equation 1).
[0217] Flow Cytometry
[0218] Flow cytometry was performed as previously described. Culture samples were collected and diluted to a final OD600 of ˜0.005-0.01 in 200 mL filtered phosphate-buffered saline supplemented with 2 mg/ml kanamycin in 96-well assay microplates (U-bottom, REF-353910 from BD Biosciences, San Jose, Calif., USA). The fluorescence of the samples was measured using a Millipore Guava EasyCyte High Throughput flow cytometer with a 488 nm excitation laser and 512/18 nm emission filter. Cytometry data was gated by forward and side scatter. FlowJo (TreeStar Inc.) was used to obtain the arithmetic mean of the fluorescence distribution. The averages of the arithmetic means were calculated from three biological replicates. The average fluorescence of the non-fluorescent wild-type cell was subtracted from each sample to obtain the final fluorescence (au) values (Equation 2). To obtain the relative fluorescence in
[0219] Hill Equation Fitting
[0220] The Hill equation (Equations 4 and 5) was used to fit lines to the fluorescence data. The model was fit to the experimentally collected data by minimizing the root mean square error (RMSE; Equation 6). Fitted values are listed in TABLE 2.
[0221] For repressible constructs:
[0222] For inducible constructs:
[0223] where
[0224] F=Calculated fluorescence
[0225] F.sub.max=Maximum fluorescence
[0226] F.sub.min=Minimum fluorescence
[0227] K.sub.A=Half maximal constant
[0228] n=Hill coefficient
[0229] [L]=Ligand concentration
[0230] where
[0231] F=Calculated fluorescence
[0232] F.sub.exp=Actual experimental fluorescence
[0233] N=Number of data points
[0234] FeaR Protein Modeling
[0235] FeaR structural motifs were annotated using the MOTIF Search web tool. Structural predictions were made using the comparative modeling function of the Robetta web server with the wild-type or mutant FeaR amino acid sequences as inputs. Predicted structures were used as the basis for ligand docking simulations. Three-dimensional conformers of the aldehydes of DA, PEA, Tyra, and Trypta were generated using Chem3D 16.0 (PerkinElmer, Waltham, Mass.). Ligand conformer and protein structure.pdb files were uploaded to the Rosetta Ligand Docking Server hosted by ROSIE, with the ligand of interest initially centered at the coordinates of the 81st residue's side chain. All protein structures were visualized using PyMOL 2.4.1 (Schrodinger, Inc., New York, N.Y.). Values for amino acid size and hydropathy index were obtained from literature for the analysis in
[0236] Quantification and Statistical Analysis
[0237] All statistical details of experiments, including significance criteria and sample size can be found in the figure legends. No sample size calculations were performed during the design of experiments. No samples were excluded.
Example 2: Engineering of a Tryptamine Biosensor
[0238] This example describes design and development of a tryptamine biosensor.
[0239] Tryptamine specific biosensors were first designed by genetic parts swapping. Three types of tynA and feaR genes from E. coli K-12 MG1655 (MG), Klebsiella aerogenes (KA), and Klebsiella pneumoniae (KP), and two types of PtynA sequences were chosen and tested in 18 different combinations (see e.g.,
TABLE-US-00011 TABLE 7 Genetic constructs used in this work. SEQ ID Recombinant NO: DNA Sequence Source 126 tynA-KA atggcaaacggcttgatgttttcccctcgtaaaaccgc Klebsiella gctggcgctggctgtcgcggtggtttgcgcctggcaat aerogenes caccggtcttcgctcacggtagcgaagcgcacatgg ATCC15038 tgccgttggataaaacgctgcaggcgttcggcgccg genome atgtgcagtgggatgactacgcgcaaatgttcaccct gataaaagacggcgcttacgtcaaagtaaaacccg gcgccaaaacggcgatcgtcaacggtaaacccctt gatctacaggtgccggtagtaatgaaggaaggtaa agcctgggtctccgatacctttatcaacgatgtattcca gtccggtctcgatcagaccttccaggtagaaaaacg ccctcacccgttaaattcgctctcggcggcggaaatc ggtgaagcagtgaccattgttaaagccgcgccgga gttccagccgaatacccgctttactgagatttccttaca cgagccggataaagcggcggtatgggcctttgccct gcagggaacacccgttgatgccccccgcaccgccg atgtggtgatgctcgatggcaaacatgtcattgaagc cgtcgtcgatctgcaaaacaaaaaaatcctctcatgg acgccgattaaaggcgcccacgggatggtcttgcttg atgacttcgtcagcgtgcagaacattatcaatgccag cagcgagttcgctgaggtgctgaaaaagcacggtat taccgaccccagtaaggtggtcaccaccccgctcac cgtcggctactttgatggcaaagatggcctgcagca ggatgcgcgtctgctgaaagtcgtcagttatctggata ccggcgacggcaactactgggcgcacccgattgaa aacctggtggcggtggtcgaccttgaagcgaagaa aatcatcaaaatcgaagaaggcccggtgatcccgg taccgatggagccacgtccgtatgatggtcgcgacc gcaacgccccggcggtgaaaccgctggagataac cgaaccggaaggcaaaaactacacgatcaccggc gataccattcactggcggaactgggattttcatctgcg cctgaactcgcgcgtcgggcccattctctcgacggtg acttacaacgacaacggcacaaaacgccaggtgat gtatgaaggttccctcggcgggatgatcgtcccctac ggcgacccggacgtcggctggtatttcaaagcctatc tggactccggcgattacggcatgggcaccctgacgt cgccgattgttcgcggtaaagatgcgccgtcaaatgc ggtactgctggacgaaactatcgccgattacaccgg caaaccgaccactattccaggcgcggtcgccatatt cgaacgctatgccgggcctgagtataagcacctgga aatgggcaaacccaacgtcagcaccgaacgccgg gaactggtggtgcgctggatcagtaccgtcgggaac tatgattatatctttgactgggtgttccatgacaacggc accatcggtatcgatgccggcgccaccggcattgaa gccgttaaaggcgtgaaggcgaagaccatgcacg accccagcgccaaagaggatacccgctacgggac gctaatcgaccataatattgtcggcaccacccacca gcatatttataatttccgectcgatctcgacgtggacgg cgaaaacaacaccctggtggcgatggatcctgaag tgaagccaaacaccgccggcggcccgcgcaccag caccatgcaggtgaatcagtacacaatcgatagcg agcagaaagcggcgcagaaattcgaccctggcact atccgcctgctgagcaacaccagcaaagagaacc gcatgggtaacccggtctcttaccagattatcccttatg ccggcggcacgcatccggcggcgaccggcgccaa gttcgccccggacgagtggatatatcatcgcctgagc tttatggataaacagctgtgggtgacgcgttaccacc cgacagagcgttatccggaagggaaatatcccaac cgttccgcccaggataccggcttaggccagtacgcg aaggatgatgagtcgctgactaaccacgacgacgt cgtgtggatcaccaccggcaccacccacgtcgcgc gcgccgaagagtggccaattatgccgaccgagtgg gcgcatgcgctgctcaaaccgtggaacttctttgacg aaaccccaacgcttggcgagaagaaaaagtaa 127 tynA-KP ATGGCAAATGGACTGAAATTCAGCC IDT CTCGTAAAACAGCTCTTGCCCTGGC CGTTGCCGTCGTTTGTGCTTGGCAA TCCCCTGTGTTTGCTCACGGTTCCG AGGCACATATGGTACCCTTGGACAA AACATTACAGGAGTTTGGGGCCGAC GTTCAGTGGGACGACTACGCACAGA TGTTTACTCTTATCAAGGACGGAGCT TATGTGAAAGTCAAGCCTGGAGCAA AAACTGCAATTGTAAATGGAAAGTCT TTAGATCTTCCCGTCCCGGTTGTGAT GAAGGAAGGCAAAGCCTGGGTTAGC GACACTTTTATCAATGATGTGTTTCA ATCCGGCCTTGATCAGACCTTTCAG GTGGAAAAGCGTCCTCATCCCTTGA ATAGTTTGAGTGCAGCCGAGATCTC TGAGGCTGTTACAATCGTTAAGGCA GCTCCGGAATTTCAACCAAATACCC GCTTTACAGAGATTTCATTGCACGAA CCGGACAAGGCTGCGGTCTGGGCT TTTGCCCTGCAAGGTACGCCAGTTG ATGCACCGCGTACCGCGGACGTCGT CATGTTAGACGGTAAGCATGTTATC GAAGCAGTAGTAGACTTGCAGAATA AAAAAATTCTTTCGTGGACTCCGATT AAGGGGGCCCACGGTATGGTCCTGT TAGACGATTTCGTCTCCGTACAAAAC ATTATCAATGCCTCATCCGAGTTTGC AGAGGTCTTGAAAAAACACGGAATT ACTGACCCGGGGAAAGTCGTGACAA CTCCCCTGACTGTAGGTTTTTTTGAT GGCAAGGATGGATTGCAACAAGACG CACGCCTGTTAAAAGTCGTCTCGTAT CTGGATACGGGTGATGGTAATTACT GGGCGCACCCTATCGAGAACTTAGT GGCCGTAGTTGACCTGGAGGCCAA GAAAATTATCAAAATCGAAGAAGGTC CAGTGATTCCCGTACCGATGGAGCC CCGTCCATACGATGGGCGCGACCG CAATGCCCCAGCTGTCAAACCTCTG GAAATCACAGAGCCTGAAGGGAAAA ATTATACTATTACTGGTGATACTATC CACTGGCAGAATTGGGACTTCCATC TGCGTCTTAATAGTCGCGTGGGACC CATTTTATCAACAGTCACTTACAATG ACAATGGGACAAAGCGTCAGGTGAT GTATGAGGGTAGTCTTGGGGGGATG ATTGTCCCCTACGGTGATCCTGATG TAGGGTGGTATTTCAAAGCTTATCTT GACTCCGGCGATTACGGCATGGGAA CGCTTACGTCGCCTATTGTTCGTGG CAAAGATGCTCCCTCGAATGCCGTT CTTTTGGATGAGACGATCGCTGATTA CACAGGCAAGCCTACTACGATTCCG GGGGCGGTGGCGATTTTTGAACGTT ACGCAGGACCTGAGTACAAACACCT TGAGATGGGAAAGCCCAATGTCAGC ACTGAACGCCGTGAGCTTGTCGTAC GTTGGATCTCAACCGTTGGTAATTAT GACTATATTTTCGACTGGGTATTTCA CGACAATGGAACGATTGGCATCGAT GCTGGGGCTACAGGGATCGAGGCA GTGAAAGGAGTGTTGGCAAAAACTA TGCATGACCCTTCGGCTAAAGAGGA TACACGCTACGGCACTCTGATTGAC CACAACATTGTGGGTACAACGCATC AGCACATCTATAACTTTCGTTTGGAC CTTGACGTTGATGGGGAGAATAACA CGCTTGTCGCGATGGACCCGGAAGT TAAGCCGAATACTGCGGGTGGTCCG CGCACGTCAACTATGCAAGTTAACC AGTACACGATCGACAGCGAGCAAAA GGCGGCTCAAAAATTCGATCCAGGA ACAATTCGTCTTTTATCTAATACCTC GAAGGAAAACCGTATGGGCAACCCG GTGTCCTACCAGATCATCCCATATG CAGGCGGTACTCACCCTGCCGCTAC CGGAGCCAAGTTTGCGCCCGACGA GTGGATTTATCACCGCCTGTCCTTTA TGGACAAACAATTATGGGTAACCCG TTACCACCCGACTGAACGCTATCCT GAAGGTAAGTACCCAAATCGCAGCG CACATGATACTGGATTAGGGCAATAT GCTAAGGATGATGAGTCATTGACTA ACCACGATGACGTTGTGTGGATCAC GACAGGAACCACTCATGTAGCGCGT GCAGAGGAGTGGCCTATTATGCCAA CGGAATGGGCGCATGCACTTCTGAA ACCTTGGAACTTTTTTGATGAGACTC CGACGTTAGGTGAAAAGAAGAAATG A 128 feaR-KA ATGGCGACAGTAGAATCCGGTCAAG IDT ATTATCAGCGCTGGTTGGCTAAGATT AACCAGGTATGCGGGCACTTCGCTG CACGCCCGCTTGGAGACGAATTTCA CGGGGAAATTGATGCGTCGTACACC GGTTCGTTGAAGGTAAGTACGGTCA CAGCACGCGGCGTAAACTTATATCG TACTCGTAACGAGATTAAACGTGACA ACGACGCATGGTTCTATACAGTGTT CCAGTTGGAAGGGCAAGCAGCTATT GAGCAGGATGAGCGCCAGGTCGCG TTAGAGGCAGGAGATATTACATTGAT CGACGCTAGTCGCCCCTGCAGTATT TTCTGGCAACACACATCTAAGCAGG CATCACTTCTTCTTCCGCGCCAATTA GTCGAGAGCCAATTGCATGGAGGAG ACATCTCTATTGCGACACGTTTGAAT AAAACGCTGCCTATGGTCCAATTAA GTCAACGTCTTCTGCAAGAATCTCTT CGCAGTCCTGCATTAAGTGAGTCGG AGGGGGAAGCGGCATTAGAGGCCA TTATCTGTCTTCTGCGCCCCATGCTG CTGCAACAAGACGCTCCGCCTACTC GCCGTGATAAACAGTTCCAAAAAATT ATGGCTTTGATTGACGAATCCATCCA ATCTGAGCAACTTCGCCCAGAATGG CTGGCGGGCGAAACAGGAATGAGT GTTCGTAGTCTGTACCGCCTGTTCG CTGATAAAGGACTTGTGGTTGCTCA GTATATTAAGAATCGCCGCTTGGATC TGTGTGCTCGTGCATTGCAGAATGC ACATGATGAAGAAAAATTGGCAGGG ATCGGGTACTCATGGGGCTTCTCAG ATCACTCGCATTTCAGCACAGCGTT CAAGCAGCGCTTCGGCGTATCCCCA GGCGAGTATCGTAAGCGTTACCGCT GA 129 feaR-KP ATGCCCGAAGCCACTCAAATGCAAC IDT AATGTGGCAATATCGTTATCACTGG GGAAGGTCGTATGATGACAACTGCG GAGGGTGGTTTAGCATATCAACGCT GGTTGGCAACCATTAACCAAGTGTG TGGTCACTTTGCTGCGCGTCCCTTA AAGGAAAGTTTCCACGGTGAAATCG ATGCTCGCTATGCGGGGTCGTTAAA GGTGTCTACCGTTACTGCTGCAGGA GTCAACCTTTATCGCACGCGTAATG AAATCAAACGCGATAACGATGCATG GTTCTATACTGTCTTCCAGTTGGCTG GCGAAGCGATCATCGAACAGGATGA TCGCCAAGTGACGCTGGCAGCAGG GGATATTACCCTTATTGACGCCGCC CGCCCATGTAGCATTGTATGGCAGC AAACATCGCGTCAAGCGTCACTTTTA TTACCCCGCCAACGTGTAGCACCGA CAGGTGATATTACAACAGCGTGTCG CTTGGACAAGAGCCTTCCTATGGTA CAACTGTCACAACGTCTTTTGTTAGA GTCGATGGGCGGAACGACTCTGTCC GCGTCAGAGTCTGAGGCGGCTCTG GAAGCAATCGCCTGCTTATTACGCC CCGTGCTTCATCAGCGTGATCCAGC GCCGAGTCGCCGTGTCAAACAGTTT CAGAAGATCATTGCGTTAATCGATG CTAGTATTCAAAGCGAACACTTGCGT CCCGAATGGTTGGCAAGCGAAACCG GAATGAGTGTACGCTCTTTATACCGT CTGTTTGCCGATCAAGGGCTGGTAG TTGCCCAGTACATCAAGAATCGCCG TCTTGACTTGTGTGCCCAAGCTCTTC AGAATGTGCACGATGACGAGAAATT AGCCGGTATTGGTTATCGTTGGGGA TTTTCTGACCATTCTCACTTCAGTAC TGCTTTCAAGCAGCGTTTCGGAGTT ACGCCCGGGCAGTACCGTAAACGCT GTCGTTAA 130 PtynA-KP/KA tatgaacgtctggcaaaactgacaaacaagctggc This work aaaagcaaacaaacccctttgcatcagtactgataa tgtgaacctgactaaaccgcccacagagcgcggttg ctaacaagaacacaacagaaagaggggttaata
[0240] Next, significant enhancement of fluorescence intensity induced by Tryptamine was shown in the engineered sensor systems containing FeaR-KA (see e.g.,