iGEM 2023 — Team ais-china

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}
![](https://static.igem.wiki/teams/4808/wiki/notebook/notebookbanner.jpg)

# Notebook

{% endblock %}

{% extends "layout.html" %}
{% block title %}Project Description{% endblock %}
{% block lead %}Description of AIS-China.{% endblock %}
{% block page\_content %}

索引

![banner](https://static.igem.wiki/teams/4808/wiki/engineeringsuccess/engineeringbanner.png)

![logo](https://static.igem.wiki/teams/4808/wiki/description/small-logo.png)

[Overview](#select-item-1)
[Engineering of the chassis
organisma](#select-item-2)
[Production of a-KB](#select-item-3)
[Further optimization.](#select-item-4)
[Temperature Control](#select-item-5)

* # Overview

  A-kb is an important molecule with potential for stimulating hair growth, naturally found in the metabolic
  pathways of various organisms. However, accumulating or extracting a sufficient amount of a-kb from organisms
  has proven challenging. Current industrial practices for a-kb production primarily rely on chemical or
  enzymatic methods, which not only incur high costs but also result in the generation of numerous harmful
  byproducts, negatively impacting the environment.

  This year, AIS-China aimed to employ synthetic biology techniques, specifically genetically modifying our
  Escherichia coli strain AIS, to produce a-kb. Our experiment was conducted in three phases. Firstly, we
  selectively knocked out three specific genes (rhtA, ilvIH, and ilvBN) in the AIS strain and increased
  expression of the desired gene ilvA, which is responsible for a-kb production. In the second phase, we
  sought to optimize the production process by introducing mutations to the ilvA gene (resulting in ilvA\*),
  utilizing vectors with different copy numbers to carry and express ilvA, and enhanced upstream genes.
  Finally, we successfully tested and implemented two temperature-sensitive repressors. We want replace the
  need for iPTG induction, thus achieving our goal of thermal-inducible expression in the future.

  In our project, we successfully achieved the accumulation of a-kb inside E. coli and attained a production
  level of 1.8 g/L. We hope for the future mass scale production of a-kb through temperature-induced
  bacteria!
  ![](https://static.igem.wiki/teams/4808/wiki/result/pic1.png)
* # Engineering of the chassis organism

  Threonine serves as the crucial precursor for a-KB production. Thus, we selected the E. coli strain CICC20905
  as the chassis organism for a-KB production, which we subsequently referred to as the AIS series, based on
  extensive research which indicated its ability to generate substantial quantities of threonine. In order to
  make our chassis organisms more advantageous, we initiated a series of genetic modifications. Initially, we
  knocked out the rhtA gene to create the AIS-1 strain. This action had the effect of retaining more threonine
  within the cell, as the rhtA gene, responsible for threonine export, was disrupted. Subsequently, base on the
  AIS-1 strains, we respectively knocked out ilvIH to construct AIS-2 and knocked out ilvBN to construct AIS-3
  strains.this would reduce the outflow of a-KB as downstream byproducts (i.e., 2-acetyl-2-Hydroxybutyrate),
  ultimately enhancing the accumulation of a-KB.

  To accomplish these genetic modifications, we constructed three pTarget plasmids, each carrying a specific
  gRNA sequence capable of identifying the target gene. We then obtained donor DNA through a two-step PCR
  process. This donor DNA was employed for homologous recombination with the genomic DNA (refer to Figure 1A).
  Subsequently, we separately introduced the pTarget plasmids along with their corresponding donor DNA into the
  host organism, which had already been engineered to carry the pEcCas plasmid. We conducted our knockout
  experiments according to the experimental procedures previously published by Qi Li, Bingbing Sun, et al. in
  2020 (as depicted in Figure 2).

  Validation of our genetic modifications was achieved through colony PCR (Figure 1B) and gene sequencing
  (Figure 1C). These analyses confirmed the successful knockout of the rhtA, ilvIH, and ilvBN genes, allowing us
  to obtain our first strains: AIS-1 (ΔrhtA), AIS-2 (ΔrhtA and ΔilvIH), and AIS-3 (ΔrhtA and ΔilvBN).Finally, we
  use three plates carrying different antibiotics to confirm the cure of pTarget and pEcCas plasmid(Figure 1D).

  ![](https://static.igem.wiki/teams/4808/wiki/result/pic2.png)

  Figure1: Knock out genes in AIS strains. （A）the design of pEcCas、pTarget plasmid and donorDNA for gene
  knockout. (B)colony PCR to respectively determine the knock-out of rhtA、ilvIH、ilvBN.（C）verified the knock-out
  result through the sequencing testing. (D)three plates carrying different antibiotics to confirm the cure of
  pTarget and pEcCas plasmid

  ![](https://static.igem.wiki/teams/4808/wiki/result/pic3.png)

  Figure 2: Step1: Transformation of the pEcCas plasmid(carry cas9 protein) into E. coli AIS-0 (CICC20905).
  Step2: construction of the pTarget plasmid and donor DNA. Step3: transformation of pTarget plasmid and donor
  DNA into the AIS-0 with pEsCas inserted. Step4: Incubation of the transformed strains.
* # Production of a-KB

  In order to produce a-KB, we sought to express the ilvA gene within the AIS strains. The ilvA gene encodes the
  enzyme Threonine Dehydrase, which is responsible for the removal of water and ammonium molecules from
  threonine (as depicted in Figure 3A). We intended to use the IPTG-induced pTac promoter system to regulate the
  expression of the ilvA gene. However, since AIS series strains of E. coli are relatively underexplored. There
  was uncertainty regarding the functionality of this system in our context.

  To ascertain whether the IPTG induction system operates effectively in AIS strains, we conducted a validation
  experiment using GFP as a signaling protein. This allowed us to assess the efficiency of the IPTG inducer and
  determine the optimal induction concentration. The results, illustrated in Figure 3B, confirmed that the AIS
  strain responded positively to the IPTG induction, with concentrations ranging from 0.1 mM to 0.2 mM
  exhibiting the strong inducing effect.In subsequent experiments, we used 0.3mM iPTG to induce gene expression,
  which is the most commonly used concentration in the laboratory.

  With the IPTG induction system established, we proceeded to construct the PW1-ilvA plasmid for a-KB
  production. This process began by obtaining the ilvA gene fragment from the E. coli genome through PCR.
  Subsequently, we integrated this fragment with the PW1 vector using the Golden Gate recombination method. The
  successful construction of the PW1-ilvA plasmid was verified through colony PCR results (see Figure 3C)
  and gene sequencing outcomes (as shown in Figure 3D).

  ![](https://static.igem.wiki/teams/4808/wiki/result/pic4.png)

  figure3: (A) ilvA function and IPTG induced-pTac promotor system.(B)test the induction sysytem by different
  iPTG concentrations in AIS strains.(C)colony PCR to determine the construction of PW1-ilvA. (D)verified the
  construction result through sequencing.

  We transformed the PW1-ilvA plasmid into strains AIS-0, AIS-1, AIS-2, and AIS-3, each with the aim of
  producing a-KB. Regrettably, our efforts to insert pw1-ilvA into AIS-3 were unsuccessful. We attribute this
  outcome to the simultaneous knockout of both the rthA and ilvBN genes, along with the overexpression of the
  ilvA gene, which appeared to hinder cell growth. We speculate that this inhibition may be due to the cytotoxic
  effects of a-KB.

  For the successfully transformed strains, we conducted HPLC methods to confirm the successful production of
  a-KB. We acquired a-KB standards from the market, by comparing the peak diagram of the standard a-KB with the
  supernatant of the AIS fermentation broth, we confirmed the production of a-Kb.Then, we made a standard curve
  with a-kb concentration of 0-20g/L in order to calculate the production of a-kb in AIS series(figure 4B).
  Observing the bar charts below, we can see that the production of a-kb increase gradually in AIS-0,AIS-1 and
  AIS-2, which indicate that the removal of rhtA gene and ilvIH gene can increase the production of a-kb (figure
  4C).

  ![](https://static.igem.wiki/teams/4808/wiki/result/chat4.jpg)

  Figure 4: (A) HPLC peak diagrams of standard a-KB and AIS supernatant sample. (B) the standard curve about the
  relationship between peak area and a-KB concentration. (C)the production of a-KB in AIS series with pw1-ilvA
  plasmid.
* # Further optimization.

  ### ilvA mutation and Copy number changed

  For further improvement on a-kb production levels, we want to optimize threonine's conversion into a-kb, a key
  step in a-kb production.We tried to use plasmids with different copy numbers to express ilvA to determine the
  effect on copy number on a-KB production. Thus, we constructed ilvA onto a new plasmid p321-ilvA, which has a
  low copy number in E. coli (Figure A)

  We also mutated ilvA(obtainning ilvA\*) to make its expressed enzymes resistant to the inhibition of
  Isoleucine. Isoleucine is a downstream product of a-kb.Our gene knock out choices do not completely obstruct
  the pathway continuing downwards of a-kb, thus meaning that Isoleucine is existent in the E.coli cell
  (Isoleucine inhibits ilvA expressed Threonine Dehydrase, thus negatively affecting Threonine's conversion into
  a-kb, which is reported in many papers).

  We made four base mutations in ilvA (i.e., C1339T, G1341T, C1351G, T1352C), which substitutes the 447th and
  451th amino acids (both Leu) into Phe and Ala, respectively (Figure B). After successfully mutating the ilvA
  gene through PCR with primers coding for the mutated sequences (Figure C), we obtained the strains pw1-ilvA\*
  and p321-ilvA\* (ilvA\* is the mutated ilvA). The two plasmids, along with pw1-ilvA and p321-ilvA, were
  transformed into AIS-2, and the a-kb production is measured. The result show that pw1-ilvA\* strain was most
  efficient. Moreover, we found that for both vectors (p321 and pw1), the mutated ilvA\* yielded more a-KB
  compared to the non-mutated ilvA, and no matter whether ilvA is mutated or not, the pw1 plasmid with higher
  copy number yielded more a-KB than the p321 plasmid with low copy number.

  ![](https://static.igem.wiki/teams/4808/wiki/result/pic6.png)

  Figure 5: (A) the different copy number of plasmid with ilvA. (B) ilvA\*'s resistancy to isoleucine inhibition
  and codon and amino acid differences between the ilvA and the mutated ilvA\*. (C) the design for the mutation
  of ilvA and the sequencing results of ilvA\*. (D)the a-kb production of strain AIS-2 with four different
  plasmid;

  ### Increase upstream concentrations:

  We have also tried modifying the upstream pathway of Threonine for more a-KB by increasing the copy number of
  gene thrABC in an effort to enhance the conversion of Aspartate into Threonine (Fig.A). For upstream
  modification, we used pcr to obtain our gene thrABC from DH5a genome, and then constructed two plasmids,
  p321-thrABC (constitutive expression) and p15a-thrABC (inducible expression). After the indication of a
  successful construction of plasmid from DNA sequencing (Fig.c), the two plasmids were each transformed into
  AIS-2, and after fermentation we measured the a-kb production. Unfortuanatley, the a-kb production did not
  improve, and instead did the contrary. This may be because, instead of an increase in threonine, an inhibition
  of the thrABC gene in the E.coli genome enhanced, in the end, leading to a decrease in production of a-kb.

  ![](https://static.igem.wiki/teams/4808/wiki/result/pic7.png)

  Figure 6: (A)the pathway of Aspartate to Threonine in E.coli. (B)Obtain gene fragments thrABC and vectors p321
  and p15A. (C) sequencing verified the construction of plasmids p321-thrABC and P15A-thrABC. (D) the a-kb
  production of AIS-2 with plasmid.
* # Temperature Control

  To enable the practical large-scale implementation of our α-kb production method, we made the strategic
  decision to transition from using IPTG-induced expression to a more user-friendly and cost-effective
  temperature-controlled induction approach. To achieve this objective,we tested two temperature sensitive
  repressors: TcI and TcI40 (TcI40 is a mutated version of TcI, shown in fig.7B).

  In comparison to TcI, TcI40 exhibited nonsynonymous mutations at six distinct positions, resulting in
  alterations to six amino acids: L65S, A67T, K68R, F115L, D126G, and D188G. Additionally, seven synonymous
  mutations (base is changed but amino acid remains constant) were also present: A50, I69, E128, R129, T152,
  S160, and L185(fig.7B).

  ![](https://static.igem.wiki/teams/4808/wiki/result/pic8.png)

  Figure 7: (A)The design of TcI-pR/pL control circuit. (B)The mutation of TcI40 compared to TcI.

  We tested the two temperature sensitive repressors in the DH5a strain with RFP as the signaling protein, and
  the test was carried out in two groups each using one of the stated repressors. The first group's RFP was
  expressed using a plasimd containing repressor TcI, and the latter group used a plamid containing repressor
  TcI40. The visible red color of expressed RFP proteins indicates if or if not the repressors loses its
  inhibitive effects on transcription (and thus expression of genes) at a given temperature.

  The results of this test show that at a temperature of 30 degrees Celsius, no visible red color can be seen
  (fig.8A), indicating that no RFP is expressed. Thus signaling that repressor TcI and TcI40 both show effective
  inhibition on the transcription process of RFP at such a given temperature. At a temperature of 37 degrees
  celcius, the transcription inhibition effect of repressor TcI ceases, and RFP expression values reaches its
  climax. At temperatures of 37, 40 and 42 degrees Celcius, the transcription inhibition effect of repressor
  TcI40 ceases, while climax values of RFP expression occur at a temperature of 40 degrees Celcius.

  ![](https://static.igem.wiki/teams/4808/wiki/result/pic9.png)

  Figure 8: Test TcI and TcI40 repressors at different induction temperatures.

  We have successfully tested two temperature control systems. By comparing TcI and TcI40 on RFP fluorescence at
  different induced temperatures, we can find that TcI40 can maximize the suppression of pR/pL promoter, so as
  to achieve the highest gene expression . In the future industrial production of a-KB, we hope to use TcI40
  repressor to induce the production of a-KB to achieve temperature control production in large scale.

  **reference：**

  Cheng L, Wang J, Zhao X, et al. An antiphage Escherichia coli mutant for higher production of L-threonine obtained by atmospheric and room temperature plasma mutagenesis. Biotechnol Prog. 2020;36(6):e3058. doi:10.1002/btpr.3058

  Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang S. A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin (Shanghai). 2021;53(5):620-627. doi:10.1093/abbs/gmab036

  Restrepo-Pineda S, O Pérez N, Valdez-Cruz NA, Trujillo-Roldán MA. Thermoinducible expression system for producing recombinant proteins in Escherichia coli: advances and insights. FEMS Microbiol Rev. 2021;45(6):fuab023. doi:10.1093/femsre/fuab023

  Chen L, Chen Z, Zheng P, Sun J, Zeng AP. Study and reengineering of the binding sites and allosteric regulation of biosynthetic threonine deaminase by isoleucine and valine in Escherichia coli. Appl Microbiol Biotechnol. 2013;97(7):2939-2949. doi:10.1007/s00253-012-4176-z

  Zhang C, Qi J, Li Y, et al. Production of α-ketobutyrate using engineered Escherichia coli via temperature shift. Biotechnol Bioeng. 2016;113(9):2054-2059. doi:10.1002/bit.25959

  Park JH, Oh JE, Lee KH, Kim JY, Lee SY. Rational design of Escherichia coli for L-isoleucine production. ACS Synth Biol. 2012;1(11):532-540. doi:10.1021/sb300071a

  Hao R, Wang S, Jin X, Yang X, Qi Q, Liang Q. Dynamic and balanced regulation of the thrABC operon gene for efficient synthesis of L-threonine. Front Bioeng Biotechnol. 2023;11:1118948. Published 2023 Mar 2. doi:10.3389/fbioe.2023.1118948

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}
![](https://static.igem.wiki/teams/4808/wiki/experiments/experimemtbanner.jpg)

# Experiments

{% endblock %}

{% extends "layout.html" %}
{% block title %}Project Description{% endblock %}
{% block lead %}Description of AIS-China.{% endblock %}
{% block page\_content %}

索引

![banner](https://static.igem.wiki/teams/4808/wiki/result/resultbanner.png)

![logo](https://static.igem.wiki/teams/4808/wiki/description/small-logo.png)

[Overview](#select-item-1)
[Engineering of the chassis
organisma](#select-item-2)
[Production of a-KB](#select-item-3)
[Further optimization.](#select-item-4)
[Temperature Control](#select-item-5)

* # Overview

  A-kb is an important molecule with potential for stimulating hair growth, naturally found in the metabolic
  pathways of various organisms. However, accumulating or extracting a sufficient amount of a-kb from organisms
  has proven challenging. Current industrial practices for a-kb production primarily rely on chemical or
  enzymatic methods, which not only incur high costs but also result in the generation of numerous harmful
  byproducts, negatively impacting the environment.

  This year, AIS-China aimed to employ synthetic biology techniques, specifically genetically modifying our
  Escherichia coli strain AIS, to produce a-kb. Our experiment was conducted in three phases. Firstly, we
  selectively knocked out three specific genes (rhtA, ilvIH, and ilvBN) in the AIS strain and increased
  expression of the desired gene ilvA, which is responsible for a-kb production. In the second phase, we
  sought to optimize the production process by introducing mutations to the ilvA gene (resulting in ilvA\*),
  utilizing vectors with different copy numbers to carry and express ilvA, and enhanced upstream genes.
  Finally, we successfully tested and implemented two temperature-sensitive repressors. We want replace the
  need for iPTG induction, thus achieving our goal of thermal-inducible expression in the future.

  In our project, we successfully achieved the accumulation of a-kb inside E. coli and attained a production
  level of 1.8 g/L. We hope for the future mass scale production of a-kb through temperature-induced
  bacteria!
  ![](https://static.igem.wiki/teams/4808/wiki/result/pic1.png)
* # Engineering of the chassis organism

  Threonine serves as the crucial precursor for a-KB production. Thus, we selected the E. coli strain CICC20905
  as the chassis organism for a-KB production, which we subsequently referred to as the AIS series, based on
  extensive research which indicated its ability to generate substantial quantities of threonine. In order to
  make our chassis organisms more advantageous, we initiated a series of genetic modifications. Initially, we
  knocked out the rhtA gene to create the AIS-1 strain. This action had the effect of retaining more threonine
  within the cell, as the rhtA gene, responsible for threonine export, was disrupted. Subsequently, base on the
  AIS-1 strains, we respectively knocked out ilvIH to construct AIS-2 and knocked out ilvBN to construct AIS-3
  strains.this would reduce the outflow of a-KB as downstream byproducts (i.e., 2-acetyl-2-Hydroxybutyrate),
  ultimately enhancing the accumulation of a-KB.

  To accomplish these genetic modifications, we constructed three pTarget plasmids, each carrying a specific
  gRNA sequence capable of identifying the target gene. We then obtained donor DNA through a two-step PCR
  process. This donor DNA was employed for homologous recombination with the genomic DNA (refer to Figure 1A).
  Subsequently, we separately introduced the pTarget plasmids along with their corresponding donor DNA into the
  host organism, which had already been engineered to carry the pEcCas plasmid. We conducted our knockout
  experiments according to the experimental procedures previously published by Qi Li, Bingbing Sun, et al. in
  2020 (as depicted in Figure 2).

  Validation of our genetic modifications was achieved through colony PCR (Figure 1B) and gene sequencing
  (Figure 1C). These analyses confirmed the successful knockout of the rhtA, ilvIH, and ilvBN genes, allowing us
  to obtain our first strains: AIS-1 (ΔrhtA), AIS-2 (ΔrhtA and ΔilvIH), and AIS-3 (ΔrhtA and ΔilvBN).Finally, we
  use three plates carrying different antibiotics to confirm the cure of pTarget and pEcCas plasmid(Figure 1D).

  ![](https://static.igem.wiki/teams/4808/wiki/result/pic2.png)

  Figure1: Knock out genes in AIS strains. （A）the design of pEcCas、pTarget plasmid and donorDNA for gene
  knockout. (B)colony PCR to respectively determine the knock-out of rhtA、ilvIH、ilvBN.（C）verified the knock-out
  result through the sequencing testing. (D)three plates carrying different antibiotics to confirm the cure of
  pTarget and pEcCas plasmid

  ![](https://static.igem.wiki/teams/4808/wiki/result/pic3.png)

  Figure 2: Step1: Transformation of the pEcCas plasmid(carry cas9 protein) into E. coli AIS-0 (CICC20905).
  Step2: construction of the pTarget plasmid and donor DNA. Step3: transformation of pTarget plasmid and donor
  DNA into the AIS-0 with pEsCas inserted. Step4: Incubation of the transformed strains.
* # Production of a-KB

  In order to produce a-KB, we sought to express the ilvA gene within the AIS strains. The ilvA gene encodes the
  enzyme Threonine Dehydrase, which is responsible for the removal of water and ammonium molecules from
  threonine (as depicted in Figure 3A). We intended to use the IPTG-induced pTac promoter system to regulate the
  expression of the ilvA gene. However, since AIS series strains of E. coli are relatively underexplored. There
  was uncertainty regarding the functionality of this system in our context.

  To ascertain whether the IPTG induction system operates effectively in AIS strains, we conducted a validation
  experiment using GFP as a signaling protein. This allowed us to assess the efficiency of the IPTG inducer and
  determine the optimal induction concentration. The results, illustrated in Figure 3B, confirmed that the AIS
  strain responded positively to the IPTG induction, with concentrations ranging from 0.1 mM to 0.2 mM
  exhibiting the strong inducing effect.In subsequent experiments, we used 0.3mM iPTG to induce gene expression,
  which is the most commonly used concentration in the laboratory.

  With the IPTG induction system established, we proceeded to construct the PW1-ilvA plasmid for a-KB
  production. This process began by obtaining the ilvA gene fragment from the E. coli genome through PCR.
  Subsequently, we integrated this fragment with the PW1 vector using the Golden Gate recombination method. The
  successful construction of the PW1-ilvA plasmid was verified through colony PCR results (see Figure 3C)
  and gene sequencing outcomes (as shown in Figure 3D).

  ![](https://static.igem.wiki/teams/4808/wiki/result/pic4.png)

  figure3: (A) ilvA function and IPTG induced-pTac promotor system.(B)test the induction sysytem by different
  iPTG concentrations in AIS strains.(C)colony PCR to determine the construction of PW1-ilvA. (D)verified the
  construction result through sequencing.

  We transformed the PW1-ilvA plasmid into strains AIS-0, AIS-1, AIS-2, and AIS-3, each with the aim of
  producing a-KB. Regrettably, our efforts to insert pw1-ilvA into AIS-3 were unsuccessful. We attribute this
  outcome to the simultaneous knockout of both the rthA and ilvBN genes, along with the overexpression of the
  ilvA gene, which appeared to hinder cell growth. We speculate that this inhibition may be due to the cytotoxic
  effects of a-KB.

  For the successfully transformed strains, we conducted HPLC methods to confirm the successful production of
  a-KB. We acquired a-KB standards from the market, by comparing the peak diagram of the standard a-KB with the
  supernatant of the AIS fermentation broth, we confirmed the production of a-Kb.Then, we made a standard curve
  with a-kb concentration of 0-20g/L in order to calculate the production of a-kb in AIS series(figure 4B).
  Observing the bar charts below, we can see that the production of a-kb increase gradually in AIS-0,AIS-1 and
  AIS-2, which indicate that the removal of rhtA gene and ilvIH gene can increase the production of a-kb (figure
  4C).

  ![](https://static.igem.wiki/teams/4808/wiki/result/chat4.jpg)

  Figure 4: (A) HPLC peak diagrams of standard a-KB and AIS supernatant sample. (B) the standard curve about the
  relationship between peak area and a-KB concentration. (C)the production of a-KB in AIS series with pw1-ilvA
  plasmid.
* # Further optimization.

  ### ilvA mutation and Copy number changed

  For further improvement on a-kb production levels, we want to optimize threonine's conversion into a-kb, a key
  step in a-kb production.We tried to use plasmids with different copy numbers to express ilvA to determine the
  effect on copy number on a-KB production. Thus, we constructed ilvA onto a new plasmid p321-ilvA, which has a
  low copy number in E. coli (Figure A)

  We also mutated ilvA(obtainning ilvA\*) to make its expressed enzymes resistant to the inhibition of
  Isoleucine. Isoleucine is a downstream product of a-kb.Our gene knock out choices do not completely obstruct
  the pathway continuing downwards of a-kb, thus meaning that Isoleucine is existent in the E.coli cell
  (Isoleucine inhibits ilvA expressed Threonine Dehydrase, thus negatively affecting Threonine's conversion into
  a-kb, which is reported in many papers).

  We made four base mutations in ilvA (i.e., C1339T, G1341T, C1351G, T1352C), which substitutes the 447th and
  451th amino acids (both Leu) into Phe and Ala, respectively (Figure B). After successfully mutating the ilvA
  gene through PCR with primers coding for the mutated sequences (Figure C), we obtained the strains pw1-ilvA\*
  and p321-ilvA\* (ilvA\* is the mutated ilvA). The two plasmids, along with pw1-ilvA and p321-ilvA, were
  transformed into AIS-2, and the a-kb production is measured. The result show that pw1-ilvA\* strain was most
  efficient. Moreover, we found that for both vectors (p321 and pw1), the mutated ilvA\* yielded more a-KB
  compared to the non-mutated ilvA, and no matter whether ilvA is mutated or not, the pw1 plasmid with higher
  copy number yielded more a-KB than the p321 plasmid with low copy number.

  ![](https://static.igem.wiki/teams/4808/wiki/result/pic6.png)

  Figure 5: (A) the different copy number of plasmid with ilvA. (B) ilvA\*'s resistancy to isoleucine inhibition
  and codon and amino acid differences between the ilvA and the mutated ilvA\*. (C) the design for the mutation
  of ilvA and the sequencing results of ilvA\*. (D)the a-kb production of strain AIS-2 with four different
  plasmid;

  ### Increase upstream concentrations:

  We have also tried modifying the upstream pathway of Threonine for more a-KB by increasing the copy number of
  gene thrABC in an effort to enhance the conversion of Aspartate into Threonine (Fig.A). For upstream
  modification, we used pcr to obtain our gene thrABC from DH5a genome, and then constructed two plasmids,
  p321-thrABC (constitutive expression) and p15a-thrABC (inducible expression). After the indication of a
  successful construction of plasmid from DNA sequencing (Fig.c), the two plasmids were each transformed into
  AIS-2, and after fermentation we measured the a-kb production. Unfortuanatley, the a-kb production did not
  improve, and instead did the contrary. This may be because, instead of an increase in threonine, an inhibition
  of the thrABC gene in the E.coli genome enhanced, in the end, leading to a decrease in production of a-kb.

  ![](https://static.igem.wiki/teams/4808/wiki/result/pic7.png)

  Figure 6: (A)the pathway of Aspartate to Threonine in E.coli. (B)Obtain gene fragments thrABC and vectors p321
  and p15A. (C) sequencing verified the construction of plasmids p321-thrABC and P15A-thrABC. (D) the a-kb
  production of AIS-2 with plasmid.
* # Temperature Control

  To enable the practical large-scale implementation of our α-kb production method, we made the strategic
  decision to transition from using IPTG-induced expression to a more user-friendly and cost-effective
  temperature-controlled induction approach. To achieve this objective,we tested two temperature sensitive
  repressors: TcI and TcI40 (TcI40 is a mutated version of TcI, shown in fig.7B).

  In comparison to TcI, TcI40 exhibited nonsynonymous mutations at six distinct positions, resulting in
  alterations to six amino acids: L65S, A67T, K68R, F115L, D126G, and D188G. Additionally, seven synonymous
  mutations (base is changed but amino acid remains constant) were also present: A50, I69, E128, R129, T152,
  S160, and L185(fig.7B).

  ![](https://static.igem.wiki/teams/4808/wiki/result/pic8.png)

  Figure 7: (A)The design of TcI-pR/pL control circuit. (B)The mutation of TcI40 compared to TcI.

  We tested the two temperature sensitive repressors in the DH5a strain with RFP as the signaling protein, and
  the test was carried out in two groups each using one of the stated repressors. The first group's RFP was
  expressed using a plasimd containing repressor TcI, and the latter group used a plamid containing repressor
  TcI40. The visible red color of expressed RFP proteins indicates if or if not the repressors loses its
  inhibitive effects on transcription (and thus expression of genes) at a given temperature.

  The results of this test show that at a temperature of 30 degrees Celsius, no visible red color can be seen
  (fig.8A), indicating that no RFP is expressed. Thus signaling that repressor TcI and TcI40 both show effective
  inhibition on the transcription process of RFP at such a given temperature. At a temperature of 37 degrees
  celcius, the transcription inhibition effect of repressor TcI ceases, and RFP expression values reaches its
  climax. At temperatures of 37, 40 and 42 degrees Celcius, the transcription inhibition effect of repressor
  TcI40 ceases, while climax values of RFP expression occur at a temperature of 40 degrees Celcius.

  ![](https://static.igem.wiki/teams/4808/wiki/result/pic9.png)

  Figure 8: Test TcI and TcI40 repressors at different induction temperatures.

  We have successfully tested two temperature control systems. By comparing TcI and TcI40 on RFP fluorescence at
  different induced temperatures, we can find that TcI40 can maximize the suppression of pR/pL promoter, so as
  to achieve the highest gene expression . In the future industrial production of a-KB, we hope to use TcI40
  repressor to induce the production of a-KB to achieve temperature control production in large scale.

  **reference：**

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  Li Q, Sun B, Chen J, Zhang Y, Jiang Y, Yang S. A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli. Acta Biochim Biophys Sin (Shanghai). 2021;53(5):620-627. doi:10.1093/abbs/gmab036

  Restrepo-Pineda S, O Pérez N, Valdez-Cruz NA, Trujillo-Roldán MA. Thermoinducible expression system for producing recombinant proteins in Escherichia coli: advances and insights. FEMS Microbiol Rev. 2021;45(6):fuab023. doi:10.1093/femsre/fuab023

  Chen L, Chen Z, Zheng P, Sun J, Zeng AP. Study and reengineering of the binding sites and allosteric regulation of biosynthetic threonine deaminase by isoleucine and valine in Escherichia coli. Appl Microbiol Biotechnol. 2013;97(7):2939-2949. doi:10.1007/s00253-012-4176-z

  Zhang C, Qi J, Li Y, et al. Production of α-ketobutyrate using engineered Escherichia coli via temperature shift. Biotechnol Bioeng. 2016;113(9):2054-2059. doi:10.1002/bit.25959

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  Hao R, Wang S, Jin X, Yang X, Qi Q, Liang Q. Dynamic and balanced regulation of the thrABC operon gene for efficient synthesis of L-threonine. Front Bioeng Biotechnol. 2023;11:1118948. Published 2023 Mar 2. doi:10.3389/fbioe.2023.1118948

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