iGEM 2021 — Team CAU_China

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# Team:CAU China/Notebook

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# Notebook

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# Team:CAU China/Engineering

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[BBa\_K3796000](#m1)
[BBa\_K3796203](#m2)
[BBa\_K3796204](#m3)

![](https://static.igem.org/mediawiki/2021/1/1a/T--CAU_China--G_13.svg)
![](https://static.igem.org/mediawiki/2021/1/1f/T--CAU_China--G_07.svg)
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# Engineering Success

CAU\_China has spent great efforts in biological engineering this year. Here we present three of our new parts that has been proved to work as desired through our engineering process, respectively [**BBa\_K3796000**](http://parts.igem.org/Part%3ABBa_K3796000), [**BBa\_K3796203**](http://parts.igem.org/Part%3ABBa_K3796203) and [**BBa\_K3796204**](http://parts.igem.org/Part%3ABBa_K3796204). You can also find more about our engineering in  [Results](https://2021.igem.org/Team%3ACAU_China/Results%20) .

### [**BBa\_K3796000**](http://parts.igem.org/Part%3ABBa_K3796000): *Corynebacterium glutamicum* strong constitutive promoter P0864

#### 1st iteration

* **Aim:** This is a constitutive promoter in *Corynebacterium glutamicum* which had little information previously on the Registry. We use this promoter to control the expression of the γ-PGA synthetase genes in our project. Therefore, we need to test its strength in *C. glutamicum* first. Also, to fully characterized this new part, we also want to know if it works as well in the typical chassis *E. coli*.

* **Design:**  We want to solve this by inserting a reporter gene behind P0864 to visually show its strength as a promoter. For convenience, we choose the fluorescent protein *mCherry*, whose excitation light and emission light both fall into the range of visible light. Also, we choose to use the shuttle vector pXMJ19 that is applicable to both *E. coli* and *C. glutamicum*. The genetic circuit we used as a device in testing is BBa\_K3796219 and the blank control is BBa\_K3796220. Particularly, you may notice that there is a tac promoter upstream of the MCS on pXMJ19, so a proper blank control to exclude the influence of Ptac leaky expression is needed. Ideally, the fluorescence intensity/OD600 should be higher in experiment group than in the control group.

  ![](https://static.igem.org/mediawiki/2021/c/c4/T--CAU_China--Results_01.svg)

  Fig.1 Genetic circuit for testing the strength of P0864

  ![](https://static.igem.org/mediawiki/2021/5/5a/T--CAU_China--Part02_mc.svg)

  Fig.2 Genetic circuit for blank control

* **Build:**  We built the genetic circuits using Clonexpress® Multi One Step Cloning kit from Vazyme to connect RBS sequence (BBa\_K3796001, synthetic) and *mCherry* gene (BBa\_J06504) downstream to P0864. We successfully constructed the above sequences and verified them by colony PCR and sequencing.

  ![](https://static.igem.org/mediawiki/2021/2/20/T--CAU_China--Results_03.png)

  **Fig. 3 Agarose gel map of BBa\_K3796219 circuit.**
  a.Lane 1 to lane 9 are electrophoresis bands of BBa\_K3796219 circuit, where lane 1 to lane 4 are for transformed *E. coli*, lane 5 to lane 9 are for transformed *C. glutamicum*. b. Lane 1 (*E. coli*) and lane 2 (*C. glutamicum*) are electrophoresis bands of BBa\_K3796220 circuit

* **Test:**  In the 1st iteration, we tested the strength of P0864 by simple qualitative test of the fluorescence under the excitation light of mCherry protein (580nm).

  ![](https://static.igem.org/mediawiki/2021/1/1c/T--CAU_China--Results_04.png)

  **Fig. 4 The difference of fluorescence in *Escherichia coli* (including experimental group (left) and control group (right)).**
  a. Image under excitation light. b. Image under natural light.

  ![](https://static.igem.org/mediawiki/2021/a/a9/T--CAU_China--Results_05.png)

  **Fig. 5 Difference of fluorescent proteins expressed by *Escherichia coli* and *Corynebacterium glutamicum*.**
  a. Images of *Corynebacterium glutamicum* after centrifugation, including experimental group (left) and control group (right). b. Images of *E. coli* after centrifugation, including experimental group (left) and control group (right).

  As you can see, compared with control group, the experiment group apparently showed red fluorescence, both under the natural light and excitation light. However, qualitative tests are not enough for the characterization of the strength of P0864, and we further wanted to know in which chassis did it work better.

* **Learn:**  We learned that P0864 could work in *E. coli* and *C. glutamicum*. The comparison of its strength between these two chassis needed to be further tested by other methods.

#### 2nd iteration

* **Re-Test:** We designed a quantitative test to better characterize this promoter in different chassis. After the bacteria had been cultured at 37 ℃ for 26 hours, the fluorescence was measured with a fluorescence spectrophotometer and OD600 was measured with a visible spectrophotometer. We use the ratio of fluorescence intensity/OD600 to reflect the relative fluorescence intensity under the unit absorbance value.

  ![](https://static.igem.org/mediawiki/2021/3/3a/T--CAU_China--Results_06.svg)

  **Fig.6 After *E. coli* and *C. glutamicum* were cultured at 37 ℃ for 26 hours, the ratio of measured fluorescence intensity data to OD600 data.**
  (the excitation light of 580nm and emission light of 610nm)

  T-tests were performed to show that there were significant difference between experiment group and control group. Plus, this difference is bigger in *C. glutamicum*.We have done four repeats and the standard deviation is acceptable.

* **Re-Learn:** P0864 is a constitutive promoter that works both in *E. coli* and *C. glutamicum*. Its strength tends to be stronger in *C. glutamicum*. If used in *E. coli* , codon-optimization may be carried out.

### [**BBa\_K3796203**](http://parts.igem.org/Part%3ABBa_K3796203): *ndoA* (endoribonuclease toxin in *Bacillus subtilis*)

#### 1st iteration

* **Aim:** We aim to test if the over-expression of the toxin gene *ndoA*, originally from *Bacillus subtilis* can kill *Corynebacterium glutamicum* effectively as well.
* **Design:**  The genetic circuit we used as a device in testing is BBa\_K3796217. We continued using the shuttle vector pXMJ19, since it has an inducible promoter, tac promoter. We inserted the gene *ndoA* between the tac promoter and rrnB terminator on the plasmid, in order to test the effect of *ndoA* with the induction of IPTG. Ideally, after adding IPTG, the growth of bacteria should be greatly depressed compared to the group without induction.

  ![](https://static.igem.org/mediawiki/2021/3/35/T--CAU_China--Results_31.svg)

  **Fig. 7 Genetic circuit for *ndoA* verification**
* **Build:**  We used ClonExpress II one-step cloning kit (Vazyme Biotech, China) to build the circuit for testing. The expression vector was transformed into *E. coli* DH5α first and then into *C. glutamicum* by electroporation. Electrophoresis showed that we had built the circuit successfully.

  ![](https://static.igem.org/mediawiki/2021/6/6c/T--CAU_China--Results_32.png)

  **Fig. 8 The agarose gel electrophoresis of enzyme-digested product. (a)(*Hin*d Ⅲ) and colony PCR (b)(*E. coli*).**.
   Lane(a): MK:Marker; 1:pXMJ19-*ndoA* digested by *Hin*d Ⅲ; Lane(b): MK:Marker; 1:Product of colony PCR (pXMJ19-*ndoA* in *E. coli*)

  ![](https://static.igem.org/mediawiki/2021/a/a6/T--CAU_China--Results_33.png)

  **Fig. 9 The agarose gel electrophoresis of colony PCR (*C. glutamicum*)**.
   Lane: MK:Marker; 1,2:product of colony PCR (pXMJ19-*ndoA* in *C. glutamicum*)(Targets are respectively endogenous *mazF* and *ndoA*); 3,4:negative control
* **Test:**  To design a quick qualitative test, ‘divided plate’ assay was carried out at the beginning. We divided a plate with LB medium into four parts and used the spread plate method to see if *ndoA* can kill *C. glutamicum* and this killing effect is not caused by the toxicity of IPTG. Bacteria carrying empty vector was added into 1 Ep tube and bacteria carrying the gene circuit was added into 2 Ep tubes with 1.8 mL LB liquid containing 10 μg/mL chloramphenicol respectively. After incubating the culture in a shaker at 30 °C, 220 rpm until OD600 reached 0.6, we prepared LB plates that were divided into four quarters marked A,B,C,D. We spread the diluted bacteria solution(10-3) on the quarters respectively, and all plates were incubated at 30 °C for a certain time.

  ![](https://static.igem.org/mediawiki/2021/3/34/T--CAU_China--Results_34.png)

  **Fig. 10 Divided Plate Assay**
  Quarter A: *C. glutamicum* carrying empty vector and 0.8mM IPTG was added; Quarter B: *C. glutamicum* carrying empty vector without adding 0.8mM IPTG; Quarter C: *C. glutamicum* carrying the vector pXMJ19-*ndoA* without adding 0.8mM IPTG; Quarter D: *C. glutamicum* carrying the vector pXMJ19-*ndoA* vector and 0.8mM IPTG was added

  Comparing Quarter A and Quarter B, we can see that the growth condition of the two is very similar, which means that the toxicity of 0.8mM IPTG is very low and it hardly kills *C. glutamicum*. Comparing Quarter C and Quarter D, we can see that there is apparently less bacteria survived in Quarter C, and the diameter of the colonies in Quarter C is rather small as well. As for the comparison between Quarter B and Quarter D, we assume that there is serious leaky expression of *ndoA* controlled by tac promoter in Quarter D, which deteriorates its growth condition. We repeated this experiments for 5 times and got nearly the same result.
* **Learn:**  We got the preliminary conclusion that *ndoA* does have a killing effect on *C. glutamicum*. However, without controlling the induction time, it’s hard to get a satisfying result because the leaky expression of the tac promoter.

#### 2nd iteration

* **Re-Test:**  To give a further quantitative test and took leaky expression into consideration, we carried out CFU assay to characterize the killing effect of ndoA instead of determining OD600 in order to get rid of dead bacterial cells. CFU was quantified by counting the colonies on one plate and normalizing the number to volume of 1 mL culture. We added 0.8mM IPTG as OD600 reached 0.6, and estimated CFU by spreading 25μL of the culture on 3 LB solid medium every hour after the induction with 0.8mM IPTG.

  ![](https://static.igem.org/mediawiki/2021/4/4e/T--CAU_China--ndoA_3_2.png)

  **Fig. 11 Results of the CFU Assay, plotted against induction time**

  It is visually discovered that the number of colonies carrying pXMJ19-ndoA fell off in the presence of IPTG, while the same bacteria grew well without induction within the first four hours and then its CFU decrease probably due to the leaky expression. We can also see that the growth condition of the bacteria carrying the empty vector hasn’t been affected by 0.8mM IPTG, since its CFU curve increases as normal. We repeated this experiments for 3 times and all the curves show similar trends.
* **Re-Learn:** We can finally come to the conclusion that the *ndoA* does have a killing effect on *C. glutamicum*, and can be used in our project as the toxin gene. In the future, we plan to change the tac promoter into a more strictly inducible one for a better characterization without leaky expression.

### [**BBa\_K3796204**](http://parts.igem.org/Part%3ABBa_K3796204): P-atp2 (alkali inducible promoter)

* **Aim:**  Patp2 is an alkali-induced promoterin our composite kill switch. We aim to verify that this promoter can respond to pH stress in *Corynebacterium glutamicum* and we also want to document its response towards different alkalinity.
* **Design:**  We want to test the strength of the promoter by reporter genes as well. Here we choose to use GFP because it is more stable to pH changes. The genetic circuit we used as a testing device is BBa\_K3796207. Particularly, we destroyed the tac promoter on the plasmid pXMJ19 to get rid of its influence this time. Ideally, the fluorescence intensity/OD600 should be higher in relatively high pH.

  ![](https://static.igem.org/mediawiki/2021/4/4c/T--CAU_China--Results_27.svg)

  **Fig. 12 Genetic circuit for Patp2 verification**
* **Build:**
  We used ClonExpress II one-step cloning kit (Vazyme Biotech, China) to build the circuit for testing. The expression vector was transformed into *E. coli* DH5α first and then into *C. glutamicum* by electroporation. Electrophoresis showed that we had built the circuit successfully.

  ![](https://static.igem.org/mediawiki/2021/7/78/T--CAU_China--Results_28.png)

  **Fig.13 The agarose gel electrophoresis of PCR product of pXMJ19-Patp2-*gfp* transformed** *E. coil*

  Lane: MK: Marker; Lane1: colony PCR product. Target: Patp2-*gfp* (825bp)

  ![](https://static.igem.org/mediawiki/2021/0/08/T--CAU_China--Results_29.png)

  **Fig. 14 The agarose gel electrophoresis of PCR product of pXMJ19-Patp2-*gfp* transformed *Corynebacterium glutamicum***

  Lane: MK: Marker; Lane1: colony PCR product. Target: Patp2-*gfp* (825bp)
* **Test:**  We continued to use the fluorescence intensity/OD600 as a measurement data. After cultivating the bacteria in the LB liquid medium at 100 rpm, 30℃ for 12h, the OD600 of the two bacteria were adjusted to nearly the same (nearly 2.0) and inoculated in the pH gradient LB liquid medium. After a cultivation of 24h, the bacteria solution was collected and washed with PBS. Then we measured its fluorescence intensity by HITACHI F-7000 according to the excitation light of 488nm and emission light of 507nm and OD600.

  ![](https://static.igem.org/mediawiki/2021/2/2a/T--CAU_China--Results_30.png)

  **Fig. 15 Fluorescence intensity results in the verification of Patp2**
  (the excitation light of 488nm and emission light of 507nm)

  Through two-way ANOVA, we can know that there’s significant difference between the control group and experiment group under different pH conditions. The relative fluorescence intensity of the control group is relatively stable at the pH range of 7-9.5, while there is a significant increase of relative fluorescence intensity in experiment group. Data shows that the difference of relative fluorescence intensity between the two groups remains high between pH=8.5 and pH=9.0, and reaches its peak near pH=9.0, indicating that the promoter Patp2 has the most ability to enhance its downstream gene expression in this pH range. Also, we can see a sharp drop of the relative fluorescence intensity in the experiment group when the pH is more than 9.5. We assume that this is because ,*C. glutamicum* can’t tolerate such a high alkalinity stress and the OD600 decreases a lot, making the relative fluorescence intensity seems abnormal or irregular. Three parallel experiments were done later, which supported our opinions.
* **Learn:**  Patp2 can respond to pH changes in *C. glutamicum*, and high alkalinity can improve the expression of the downstream genes of Patp2. According to our data, the peak occurs near pH=9.0, which happens to fall in the range of the alkalinity of saline-alkaline soil. The strength pf Patp2 under higher pH needs to be further tested.

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# Team:CAU China/Experiments

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# Protocols

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# Team:CAU China/Results

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# Results

## Part I: Make *Corynebacterium glutamicum* produce polyglutamic acid, and measure the expression of P0864 promoter.

## Work we have finished

### 1.Determination of constitutive promoter (P0864(BBa\_K3796000))strength

P0864(BBa\_K3796000) is a constitutive promoter in *Corynebacterium glutamicum* which had little information previously on the Registry. We use this promoter to control the expression of the γ-PGA synthetase genes in our project. Therefore, we need to test its strength in *C. glutamicum* first. Also, to fully characterize this new part, we want to know whether it works in the typical chassis *E. coli* as well.

We want to achieve this goal by inserting a reporter gene after P0864 to visually show its strength as a promoter. For convenience, we choose the fluorescent protein mCherry, whose excitation light and emission light both fall into the range of visible light. Also, we choose to use the shuttle vector pXMJ19 that is applicable to both *E. coli* and *C. glutamicum*. The genetic circuit we use as a device in testing is BBa\_K3796219 and the blank control is BBa\_K3796220. Particularly, you may notice that there is a tac promoter upstream of the MCS on pXMJ19, so a proper blank control to exclude the influence of Ptac leaky expression is needed. Ideally, the fluorescence intensity/OD600 should be higher in the experimental group than in the control group.

![lacIq-Ptac-P0864-RBS-mcherry-rrnB](https://static.igem.org/mediawiki/2021/c/c4/T--CAU_China--Results_01.svg)

**Figure 1 Genetic circuit for testing the strength of P0864**

![lacIq-Ptac-RBS-mcherry-rrnB](https://static.igem.org/mediawiki/2021/5/5a/T--CAU_China--Part02_mc.svg)

**Figure 2 Genetic circuit for blank control**

We built the genetic circuits using Clonexpress® Multi One Step Cloning kit from Vazyme to connect RBS sequence (BBa\_K3796001, synthetic) and mCherry gene (BBa\_J06504) downstream to P0864. We successfully constructed the above sequences and verified them by colony PCR and sequencing.

![A:Validation of E. coli transformants(BBa_K3796219) by colony PCR;B:Validation of C. glutamicum transformants(BBa_K3796219) by colony PCR ](https://static.igem.org/mediawiki/2021/2/20/T--CAU_China--Results_03.png)

**Figure 3 Agarose gel map of BBa\_K3796219 circuit.**

a.Lane 1 to lane 9 are electrophoresis bands of BBa\_K3796219 circuit, where lane 1 to lane 4 refer to transformed *E. coli*, lane 5 to lane 9 refer to transformed *C. glutamicum*. b. Lane 1 (*E. coli*) and lane 2 (*C. glutamicum*) are electrophoresis bands of BBa\_K3796220 circuit

In the 1st iteration, we tested the strength of P0864 by a simple qualitative test of the fluorescence under the excitation light of mCherry protein (580nm).

![The difference of fluorescence in Escherichia coli ](https://static.igem.org/mediawiki/2021/1/1c/T--CAU_China--Results_04.png)

**Figure 4 The difference of fluorescence in *Escherichia coli* (including experimental group (left) and control group (right)).**

a. Image under excitation light. b. Image under natural light.

![Difference of fluorescent proteins expressed by Escherichia coli and Corynebacterium glutamicum.](https://static.igem.org/mediawiki/2021/a/a9/T--CAU_China--Results_05.png)

**Figure 5 Difference of fluorescent proteins expressed by *Escherichia coli* and *Corynebacterium glutamicum*.**

a. Images of *Corynebacterium glutamicum* after centrifugation, including experimental group (left) and control group (right). b. Images of *E. coli* after centrifugation, including experimental group (left) and control group (right).

As you can see, compared with the control group, the experimental group showed red fluorescence apparently, both under the natural light and excitation light. However, qualitative tests are not enough for the characterization of the strength of P0864, and we further wanted to know in which chassis did it work better.

We designed a qualitative test to better characterize this promoter in different chassis. After the bacteria had been cultured at 37 ℃ for 26 hours, the fluorescence was measured with a fluorescence spectrophotometer and OD600 was measured with a visible spectrophotometer. We use the ratio of fluorescence intensity/OD600 to reflect the relative fluorescence intensity under the unit absorbance value.

![ After E. coli and C. glutamicum were cultured at 37 ℃ for 26 hours, the ratio of measured fluorescence intensity data to OD600 data.](https://static.igem.org/mediawiki/2021/3/3a/T--CAU_China--Results_06.svg)

**Figure 6 After *E. coli* and *C. glutamicum* were cultured at 37 ℃ for 26 hours, the ratio of measured fluorescence intensity data to OD600 data.**
(the excitation light of 580nm and emission light of 610nm)

T-tests were performed to show that there was a significant difference between the experimental group and the control group. Plus, this difference is bigger in *C. glutamicum*. We have done four repeats and the standard deviation is acceptable.

We learned P0864 is a constitutive promoter that works both in *E. coli* and *C. glutamicum*. Its strength tends to be stronger in *C. glutamicum*. If used in *E. coli*, codon-optimization may be carried out.

### 2. Modular construction of *capA* *capB* and *capC*

For each gene in the *capABC* gene cluster, we connected P0864, RBS and terminator sequences upstream and downstream of them.

![a. capA module:P0864-RBS-capA-T7t;b.capB module:P0864-RBS-capB-T7t;c.capC module:P0864-RBS-capC-T7t](https://static.igem.org/mediawiki/2021/1/13/T--CAU_China--Results_07.png)

Because the length of the module is large, and the head and tail sequences of the three fragments are the same, we designed different methods to add specific marker sequences to the head and tail, and tried to integrate the *capA* module, *capB* module and *capC* module by seamless cloning or enzyme digestion, but all failed.

![](https://static.igem.org/mediawiki/2021/a/ab/T--CAU_China--Results_09.png)

**Figure 9 The agarose gel electrophoresis of PCR(*capC*)**
We amplified the *capC* module to test the connection of the three modules.

We tested the transformed bacteria (*E. coli* DH5α) and the colony PCR verification was carried out. The target band shown in the figure was the *capC* module. In the detection, 12 plaque bands could be amplified, of which 6 bands were obviously bright. However, we could not extract the corresponding plasmids from these bacteria.

## Conclusion

At present, we have completed the determination of P0864 expression in *E. coli*. However, it was not successfully transformed into *Corynebacterium glutamicum*.

We completed the construction of each gene module of *capABC*, but failed to connect the three modules.

## Failures & Difficulties

In the experiment, we encountered many difficulties in construction and detection, as well as the difficulties from COVID-19. Further, our experiment was short of funds. In this case, many kits could not be purchased in time. Therefore, we summarized a relatively simple identification process after recombinant plasmid transformation: colony PCR → Plasmid Extraction → Sanger sequencing (test pass or one-way sequencing). We omitted the step of enzyme digestion verification, which consumed a large amount of enzyme and could not avoid the step of Sanger sequencing. The companies that cooperate with us in sequencing include Sangon and Tsingke. In addition, in the case of lack of funds, we also reduced the amount of nucleic acid dye as much as possible. Each time we used agarose gel, we used 20ml 1× TAE buffer, add 2 μL nucleic acid dye. Nucleic acid dyes still have good dyeing ability at this dosage, and can significantly reduce the dosage.

## Part II: Transformation of *Corynebacterium glutamicum* metabolic pathway. The expression of *ppc* and *odhA* was enhanced and decreased α-Ketoglutarate dehydrogenase expression

## Work finished

### 1.*odhA*

In order to improve the production of glutamic acid which is formed from the citric acid cycle intermediate α-ketoglutarate, we decided to attenuate *odhA* which encodes KGDH that catalyzes α-KG to form succinyl-CoA formation to introduce more carbon fluxes to glutamic acid synthesis rather than to the succinyl-CoA formation. So we designed the experiments of introducing a weaker strength RBS to the upstream of its ORF for *odhA* attenuation.

![upstream homologous arm-Strong strength RBS-ppc-downstream homologous arm](https://static.igem.org/mediawiki/2021/8/85/T--CAU_China--Results_10.svg)

**Figure 10 Sequence for modifying RBS upstream of *ppc* in *C. glutamicum* genome**

#### Construction of Recombinant Plasmid pK18mob*sacB*-RBS-*odhA*

The first thing to introduce a weaker strength RBS to the upstream of its ORF is constructing the recombinant plasmid pK18mob*sacB*-RBS-*odhA* successfully. The weak RBS addition step was completed in the *odhA* amplification stage. To achieve this goal, we decided to amplify *odhA* by dividing it into *odhA*-UP and *odhA*-DOWN to amplify.

![ The agarose gel electrophoresis of PCR (odhA-UP) Lane ](https://static.igem.org/mediawiki/2021/5/5a/T--CAU_China--Results_11.png)

**Figure 11 The agarose gel electrophoresis of PCR (*odhA*-UP) Lane MK: Marker; Lane1: the target band of *odhA*-UP**

![The agarose gel electrophoresis of PCR(odhA-DOWN)](https://static.igem.org/mediawiki/2021/1/1b/T--CAU_China--Results_12.png)

**Figure 12 The agarose gel electrophoresis of PCR(*odhA*-DOWN) Lane MK: Marker; Lane1: the target band of *odhA*- DOWN**

After amplification of *odhA*-UP and *odhA*-DOWN, we digested pK18mob*sacB* by *Sma*I, and fused *odhA*-UP, *odhA*-DOWN and *Sma*I-pK18mob*sacB* to construct the combinate plasmid by One Step Cloning Kit---ClonExpressTMMultiS. Then the combination plasmid was transferred into DH5α for plasmid amplification.

In order to identify whether the combination plasmid was constructed successfully or not, we used three ways to verify it. The first method was colony PCR whose template was the exposed DNA after cell pyrolysis (Figure 4). Secondly, through enzyme digestion verification we observed the target (Figure 5). Finally, we extracted the plasmids in DH5α, sequenced them and the response was correct.

![The agarose gel electrophoresis of colony PCR to verify recombinant plasmid](https://static.igem.org/mediawiki/2021/e/e0/T--CAU_China--Results_13.png)

**Figure 13 The agarose gel electrophoresis of colony PCR to verify recombinant plasmid(pK18mob*sacB*-RBS-*odhA*)**

![Enzyme digestion verification(pK18mobsacB-RBS-odhA SmaI)](https://static.igem.org/mediawiki/2021/1/18/T--CAU_China--Results_14.png)

**Figure 14 The agarose gel electrophoresis of the enzyme-digested product(*Sma*I)**

Lane MK: Marker; Lane1: the band of pK18mob*sacB*-RBS-*odhA*; Lane1: the target band of pK18mob*sacB*-RBS-*odhA* disgested with *Sma*I

#### Transformation of *Corynebacterium glutamicum* ATCC 13032

The following step after successfully constructing the pK18mob*sacB*-RBS-*odhA* combination plasmid was to transform it into *Corynebacterium glutamicum* ATCC 13032 by electroporation (Figure 6). Because pK18mob*sacB* is a mobilizable vector with KmR and *sacB* allowing for selection of double crossover in *C. glutamicum*, we default *C. glutamicum* has occurred the first exchange if it can grow on the LBG-Kan solid medium. In order to further verify the first exchange has already happened, we performed colony PCR validation, and the result displayed that the 9th strain has the target fragment.

![Transformants of Corynebacterium glutamicum ATCC 13032(pK18mobsacB-RBS-odhA)](https://static.igem.org/mediawiki/2021/3/32/T--CAU_China--Results_15.png)

**Figure 15 Transformation of *Corynebacterium glutamicum* ATCC 13032**

![The agarose gel electrophoresis of colony PCR to verify the first exchange.](https://static.igem.org/mediawiki/2021/6/60/T--CAU_China--Results_16.png)

**Figure 16 The agarose gel electrophoresis of colony PCR to verify the first exchange.**
Lane MK: Marker; Lane1: positive control; Lane2: the target band

### 2.*ppc*

In order to construct a strain with high glutamic acid yielding, we have decided to achieve this goal through the double-crossover replacement technique with plasmid pK18mob*sacB*, a plasmid containing both kanamycin resistance and *sacB* for double screening. PEPC encoded by *ppc* in the TCA cycle catalyzes PEP to form OAA. By overexpressing *ppc* through an RBS replacement, we expect a growth in glutamic acid production in our recombinant strain.

We have constructed a recombinant plasmid pK18mob*sacB*-RBS-*ppc* for the RBS replacement in *Corynebacterium glutamicum* ATCC 13032, the results are as follows.

![PCR for ppc-UP  .](https://static.igem.org/mediawiki/2021/4/4b/T--CAU_China--Results_17.png)

**Figure 17 Amplification of *ppc*-UP.**
Lane: MK: Maker; Lane1: *ppc*-UP.

![PCR for ppc-DOWN  .](https://static.igem.org/mediawiki/2021/b/be/T--CAU_China--Results_18.png)

**Figure 18 Amplification of *ppc*-DOWN.**

MK: Maker; Lane1: *ppc*-DOWN.

![Restriction digestion of pK18mobsacB with SmaI . ](https://static.igem.org/mediawiki/2021/5/5f/T--CAU_China--Results_19.png)

**Figure 19 Restriction digestion of pK18mob*sacB* with *Sma*I.**

MK: Maker; Lane1: *Sma*I-pK18mob*sacB*.

![Colony PCR validation  ](https://static.igem.org/mediawiki/2021/0/01/T--CAU_China--Results_20.png)

**Figure 20 Colony PCR.**
Lane: MK: Maker; Lane1: positive control; Lane2: colony PCR product.

![Double digestion with XbaI and EcoRV (pK18mobsacB)  .](https://static.igem.org/mediawiki/2021/0/04/T--CAU_China--Results_21.png)

**Figure 21 Double digestion with *Xba*I and *Eco*RV.**

MK: Maker; Lane1: pK18mob*sacB*-RBS-*ppc*; Lane2: double-digested product.

What’s more, we further delivered the plasmid from positive strains selected above to sequencing and were able to confirm the correct target plasmid.

## Work unfinished

### 1.*odhA*

#### Verification of the second exchange of double crossing

After verifying the first exchange has occurred, the 9th strain was cultured in the LBG solid medium to induce the second exchange, but when we verified by using resistance screening, it failed.

#### Phenotypic verification

We plan to do the activity test of enzyme ODHA and the glutamic acid production test to verify our experiment-*odhA* attenuation.

### 2.*ppc*

With the successful construction of recombinant plasmid pK18mob*sacB*-RBS-*ppc*, we then attempted to transform it into the competent cells of *Corynebacterium glutamicum* ATCC 13032. However, we have not screened any positively recombinant strains yet. For the future, if we could successfully construct a recombinant strain with the RBS change in *Corynebacterium glutamicum* ATCC 13032, we would be able to carry out measurement of enzyme activity and glutamic acid production assay.

### 3.RNAi

We plan to reduce enzyme activity by RNAi technology, prevent the degradation of γPGA by bacteria, and finally achieve the goal of producing γPGA.

![lacIq-Ptac-anti odhA-rrnB](https://static.igem.org/mediawiki/2021/6/6c/T--CAU_China--Results_22.svg)

**Figure 22 RNAi circuit**

Construction method:

By designing a suitable primer, the anti enzyme DNA sequence with RBS is amplified from the *Corynebacterium glutamicum*. The sequence is connected to the plasmid by Restriction enzyme digestion, and the recombinant plasmid is imported into *E. coli* to verify the success of plasmid construction. If successfully constructed, the recombinant plasmids will be imported into *Corynebacterium glutamicum* to complete the transformation of engineering bacteria.

## Conclusion

### 1.*odhA*

a) We first completed the experimental design of this program. Then, through the technical means of molecular biology, we completed the construction of the recombinant plasmid pK18mobsacB-RBS-*odhA*. After that, we carried out the transformation of *Corynebacterium glutamicum* ATCC 13032.

b) In order to truly complete the transformation of glutamic acid to increase production, in the future, we will perform phenotypic verification by the activity test of enzyme ODHA and perform the glutamic acid production test to ensure that the plasmid we introduced really plays a role in *Corynebacterium glutamicum*.

### 2.*ppc*

With the successful construction of recombinant plasmid pK18*mobsacB*-RBS-*ppc*, we then attempted to transform it into the competent cells of *Corynebacterium glutamicum* ATCC 13032. However, we have not screened any positively recombinant strains yet. For the future, if we could successfully construct a recombinant strain with the RBS change in *Corynebacterium glutamicum* ATCC 13032, we would be able to carry out the measurement of enzyme activity and glutamic acid production assay.

### 3.RNAi

The first round of construction is currently under way after the introduction of *E. coli* electrophoresis verification failed, and the second round of rebuilding is under way. The second round of builds is expected to be completed by the deadline.

In the future, we need to be determined to build successfully on the basis of the anti sequence into *Corynebacterium glutamicum*, and the gene is integrated into the genome so that the interference effect can be stable in a long term.

## Failure & difficulties

### 1.*odhA*

* The effect of PCR under theoretical conditions is not up to expectations. Through gradient PCR, we have improved the PCR conditions for extracting target fragments to ensure that sufficient target fragments are obtained.
* Seamless cloning and electrotransfer require multiple attempts before success.
* The selection of homologous recombination steps has been adjusted many times. For example, the initial scheme was positive screening and negative screening. After several experiments failed, after discussion, we adjusted the screening method to relaxation culture.

### 2.*ppc*

At first, our amplification of *ppc*-UP and *ppc*-DOWN failed. Considering the high GC content of our target strain, we changed the primers and conducted gradient PCR to determine the proper annealing temperatures, and finally managed to overcome the setbacks.

The transformation of *Corynebacterium glutamicum* ATCC 13032 failed. Furthermore, another successfully constructed recombinant plasmid using the backbone of pK18*mobsacB* was not able to transform *Corynebacterium glutamicum* ATCC 13032 either, while constructions using the backbone of pXMJ19 succeeded. We are not sure that if the problem is caused by the backbone plasmid yet, but we hope to conduct more experiments by changing the backbone for another round of construction and so on in the future.

### 3.RNAi

The RBS area was not designed during the first design sequence, resulting in ineffective previous experimental work. It remind us to think hard before building a target fragment, and make sure the experiment design is correct before experimenting.

## Part III: Kill switch construction

## Work Finished:

### 1.Verified that PgsiB can respond to salinity changes in *C. glutamicum*

We aim to verify that the promoter PgsiB, originally from *Bacillus subtilis*, can respond to salt stress in *Corynebacterium glutamicum* and we also want to document its response towards different salt concentrations.

![PgisB-RBS-gfp-rrnB](https://static.igem.org/mediawiki/2021/3/35/T--CAU_China--Results_23.svg)

**Figure 23 Gene circuit for PgsiB verification**

To characterize its response, we built a plasmid pXMJ19-PgsiB-*gfp* using the ClonExpress II one-step cloning kit (Vazyme Biotech, China) in order to test the function of PgsiB by the fluorescence of GFP. Particularly, we destroyed the tac promoter on the plasmid pXMJ19 to get rid of its influence.

![The agarose gel electrophoresis of PCR product of pXMJ19-PgsiB-gfp transformed E. coli Target:818bp.](https://static.igem.org/mediawiki/2021/1/12/T--CAU_China--Results_24.png)