iGEM 2023 — Team buct

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July 21

- Successfully extracted gltAB (pstar HS) from the genome of Pseudomonas aeruginosa, and it will be
  amplified again. Enzymatic digestion and transformation coating are scheduled for tomorrow.
- Extracted the genomes of both the first and second generations of Legionella pneumophila. PCR will
  be
  performed once the primers arrive tomorrow.

- gltAB: Enzymatic digestion, ligation, and transformation coating have been performed. Tomorrow, the
  growth status of the bacteria will be observed for verification, and they will be transferred to
  test
  tubes.
- pgsBCA: After re-ligation and transformation yesterday, a total of 7 bacterial colonies have grown.
  After
  bacterial verification, there were no issues, and they have been transferred to culture tubes.
- ureABC: Utilizing primers from the literature, ureABC has been successfully obtained from the
  genomes of
  both the first and second generations of Legionella pneumophila that were extracted earlier. It has
  been
  collected and will undergo enzymatic digestion tomorrow.

July 22

July 24

- The pmv plasmid containing the racE gene (already extracted) has been purified. The racE gene has
  been
  connected using Gibson assembly and then subjected to transformation.
- Similarly, ureABC and gltAB have been connected using redesigned primers via Gibson assembly and
  subsequently transformed.

- PMV-racE was extracted, and racE ureABC gltAB were obtained through Gibson primer PCR. Using Gibson
  assembly, we constructed four plasmids: petlac-ureABC, pcs-gltAB, pcs-racE, and petlac-ureABC
  connected
  with T4. Transformation and coating processes were successfully completed.
- Experimental Team Members: Yu Linwei, Liu Yazhao, Xie Wenjun.

July 25

July 26

- Eight individual colonies from each of the ureABCx2, gltAB, and racE plates that were transformed
  yesterday were selected.
- After bacterial verification, the correct strains were transferred into test tubes. Plasmid
  extraction
  and subsequent uniform sequencing are planned.

- Up to this point, all required plasmids have been successfully constructed (petlac-ureABC,
  petlac-pgsBCA,
  pcs-racE, pcs-gltAB, pcs-creJ, totaling five types, with a total of 12 plasmids). They have been
  sent
  for
  sequencing by the company. If there are any sequencing errors, adjustments will be made accordingly.
- For gltBD and glnA, which are the parts needed for CRISPR integration, genes have been extracted
  from the
  BW25113 genome. This yielded ygay up and ygaydown, two homologous arm segments, as well as the glnA
  and
  50xtrc promoter parts. However, obtaining gltBD has proven challenging, and we are currently in the
  process
  of adjusting the plan.

July 28

August 4

- The glutamate synthesis pathway has been changed to GDH, and the desired integration fragment has
  been
  successfully constructed. It will be electroporated tomorrow.
- All plasmids constructed previously have been sequenced correctly. Next steps involve changing the
  required
  promoters and separating and integrating different expression frames onto the designed plasmids.

- The pcs-gltAB plasmid was extracted, and the expression frame and backbone were reverse cloned.
  Simultaneously, the creJ expression frame was cloned out. After homologous recombination of these
  two,
  transformation coating was performed.
- The integrated bacteria were selected and subjected to bacterial verification. Once confirmed to be
  error-free, they were transferred to A+Sp medium.

August 10

August 17

- Constructed PETlac-pgsBCAE, pCS-lac-racE, and pCSlac-gltAB plasmids.
- Transformed these plasmids into our previously prepared host bacteria using electroporation and plated them for overnight cultivation.

- Retrieved plated bacteria from the previous day and inoculated single colonies into test tubes for subsequent shaking flask fermentation.
- Prepared substrate solutions required for fermentation.

August 18

August 19

- Conducted fermentation verification for the first two modules and included a blank control group.

- Completed the first batch of fermentation and collected samples for subsequent liquid-phase measurements.
- Constructed all elements for sub-module validation.

August 22

August 28

- Constructed PETduet-ureABC-lac-EFGD and, according to literature data, used BL21 as the vector to observe its inclusion bodies. We attempted to address the solubility issue of urease originating from Providencia species in the EcN vector.

- Purified the fermented urease protein and characterized its solubility through SDS-PAGE electrophoresis.

August 31

September 4

- Constructed pze-glnAP2-mcherry and attempted to induce glnAP2 expression under nitrogen-deficient conditions.
- Created a random mutation library of glnAP2 promoter through error-prone PCR.

- Designed experiments to integrate all three modules into one host bacterium.
- Constructed pETlac-ureABC-plpp1.2-ureEFGD-lac-pgsBCAE and pZE-glnAp2-ttgR-asrG-Pttg-bsrG plasmids.

September 10

September 12

- Transformed pETlac-ureABC-plpp1.2-ureEFGD-lac-pgsBCAE and pZE-glnAp2-ttgR-asrG-Pttg-bsrG plasmids into host bacteria and plated for antibiotic resistance screening.

- Successfully cultured host bacteria containing pETlac-ureABC-plpp1.2-ureEFGD-lac-pgsBCAE and pZE-glnAp2-ttgR-asrG-Pttg-bsrG plasmids.
- Conducted fermentation validation of the entire synthetic pathway we designed.

September 14

September 15

- Transformed host bacteria with the original plasmid pze-glnAP2-mcherry without a random mutation of the glnAP2 promoter.

- Inoculated strains containing the pze-glnAP2-mcherry plasmid into test tubes for overnight cultivation.

September 16

September 17

- Transferred strains containing the pze-glnAP2-mcherry plasmid into a 96-well plate with a series of ammonia concentration gradients.
- After 24 hours of cultivation, measured the intensity of red fluorescent protein to characterize the sensitivity and detection limit of the glnAP2 promoter response to ammonia.

- Transformed plasmid pze-glnAP2-random-mcherry with a random mutation of the glnAP2 promoter into host bacteria.

September 21

September 22

- Inoculated 96 strains containing pze-glnAP2-random-mcherry plasmid into a 96-well plate for cultivation.

- Transferred strains containing pze-glnAP2-random-mcherry plasmid into a 96-well plate with ammonia concentration gradients as per our experiment's settings.
- After 24 hours of cultivation, measured the intensity of red fluorescent protein to conduct targeted screening of the random mutation.

September 23

October 1

- Successfully obtained four random mutation promoters based on repeated experiments.
- Designed a suicide system.

- Began constructing pZE-glnAp2-ttgR-asrG-Pttg-bsrG plasmid.

October 2

October 6

- Successfully constructed the pZE-glnAp2-ttgR-asrG-Pttg-bsrG plasmid.
- Electroporated it into host bacteria for subsequent characterization.

- Picked plated bacteria containing the pZE-glnAp2-ttgR-asrG-Pttg-bsrG plasmid and inoculated them into test tubes with different ammonia concentrations for overnight cultivation.

October 7

October 8

- Measured the OD of bacterial cultures at each ammonia concentration to validate our designed suicide system.

- Completed all experiments for the project.

October 10

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{% block title %}Engineering success{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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* CONTENTS
* [Degradation of creatinine](#part1)
* [Degradation of urea](#part2)
* [Synthesis of polyglutamic acid & a complete pathway](#part3)
* [Suicide system](#part4)

BUCT pursues preciseness and efficiency in the process of exploring problems. We are not afraid of failure. After experiencing failure, we will continue to review, discuss, draw lessons and strive for higher achievements!

BUCT's exploration process for problems can be roughly summarized into three cycles, cycle 1, 2 and 3. In each cycle, we will continue to move forward in the way of try face the problem solve the problem further try.

## Degradation of creatinine

| Cycle 1 | Cycle 2 | Cycle 3 |
| --- | --- | --- |
| We used Pseudomonas putida NBRC 14164 preserved in the laboratory for resuscitation culture, and extracted the whole genome of the bacterium. It was found that the fragment could not be amplified by conventional methods. We found that the reason might be the inappropriate annealing temperature. Therefore, we tried to use the method of cyclic heating up. After continuous attempts, we amplified crnA by touch down PCR, and ordered the gene fragment of crej, After homologous recombination on pCS27-lac plasmid, pCS27-lac-crnA and pCS27-lac-crej-were obtained, which were correct after sequencing. | The expression frame in pCS27-lac-crnA was amplified separately (with SacI and BcuI digestion sites) and connected with the digested pCS27-lac-creJ under the action of T4 ligase, but the transformation efficiency was not high. Therefore, we amplified the two expression frames separately, connected them by overlap, homologous recombination with the pCS27 plasmid bone frame, and transformed them after sequencing without error. The fermentation experiment was carried out, and good results were obtained. | After the effect of creatinine degradation was successfully verified, considering that crnA and creJ have different reaction rates, we combined them into an expression box and adjusted their arrangement order. In order to reduce the burden of bacteria, we integrated the glutamate racemase coding gene with the same expression intensity into the same plasmid to construct a more complex coexpression plasmid pCS27-lac-crej-crnA race. We learned from the previous lessons, connected each expression frame by overlap, and then homologous recombination with the plasmid backbone. Finally, we sequenced the obtained plasmid, and the results were correct. |

## Degradation of urea

| Cycle 1 | Cycle 2 | Cycle 3 |
| --- | --- | --- |
 We first purchased Provincia rettgeri strain jnb815 from CICC and obtained its whole genome. The urease gene cluster was obtained through the primer sequences provided in the corresponding literature. Through data investigation, we found that ureEFGD, an auxiliary gene of urease, is not soluble. Therefore, we tried to solve or improve this problem. First, we linked it to pET duet plasmid, transformed it into E. coli BL21 to induce protein production, and then broken it. SDS-PAGE verified that groES/EL has a certain effect. | We constructed pETlac-ureABC-lac-ureEFGD and carried out the addition experiment of urea degradation, but the results were not ideal. In order to enhance the effect of ureEFGD, we replaced the lac promoter, so we constructed pze12-plpp1.2-ureEFGD, amplified its expression frame, and constructed pETlac-ureABC-plpp1.2-ureEFGD. The fermentation experiment was carried out again, and the results were slightly improved. | Similarly, in order to reduce the burden of bacteria, we integrated polyglutamic acid synthase with the above modules, and finally constructed a more complex coexpression system pETlac-ureABC-plpp1.2-ureEFGD-lac-pgsBCAE |

## Synthesis of polyglutamic acid & a complete pathway

| Cycle 1 | Cycle 2 | Cycle 3 |
| --- | --- | --- |
| First of all, we need to enhance the precursor supply of glutamate, the substrate of this process. We obtained the coding gene of glutamate dehydrogenase in the genome of E. coli K12, and used crispr-cas9 technology at the ygay site to integrate the genome of EcN. However, when the pcas9 plasmid was eliminated, it could not be effectively eliminated at 42 ℃ for 12h. Therefore, we extended the time to 24h and achieved success.  After that, we ordered the gene sequence of glutamate racemase (race) and obtained the gene cluster pgsBCAE of polyglutamic acid synthase in the genome of Bacillus subtilis strain a-5, and constructed pETlac-pgsBCAE, pCS27 lac race. After sequencing, it was correct. | The above two plasmids were co transformed into the chassis to carry out the fermentation experiment of glutamate addition (because the work of gene integration was not completed at this time), and good results were obtained. | In the verification of the complete pathway, we constructed two more complex coexpression plasmids pCS27-lac-crej-crnA-lac-race, pETlac-ureABC-plpp1.2-ureEFGD-lac-pgsBCAE, and completed the verification of fermentation with gdhA integrated Escherichia coli Nissle 1917 as the chassis. |

## Suicide system

| Cycle 1 | Cycle 2 | Cycle 3 |
| --- | --- | --- |
| Our initial idea was to build a temperature sensing suicide system, using ci857 as a key element, but after our discussion, this element lacked enough innovation to help future teams, so we abandoned it.  Later, we decided to use the difference of ammonia nitrogen concentration to build an innovative biosafety system. We localized glnAp2. After our characterization, the promoter can show different expression intensity due to the change of external ammonia nitrogen concentration, but the intensity change it shows cannot meet our actual needs. Therefore, mutation of this sequence has become our main task. | Fortunately，we screened about 1600 samples in total, and finally found these four samples in all mutated samples, which is a considerable workload for BUCT, which is completely composed of undergraduate students. After obtaining the correct mutated samples, we sequenced them. | We successfully used this element to construct a suicide system with bsrg/sr4 as toxin antitoxin and ttgr PTTG as repression system (we obtained them in the genomes of Bacillus subtilis and Pseudomonas putida), and verified the feasibility of the suicide system under the induction of different concentrations of ammonia nitrogen. |

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{% block title %}Results{% endblock %}
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* CONTENTS
* [Creatinine degradation module](#part1)
* [● Gene cloning](#part2)
* [● Co-expression plasmid construction](#part3)
* [● Fermentation Additive Experiment](#part4)
* [Urea degradation module](#part5)
* [● Gene cloning](#part6)
* [● Co-expression plasmid construction](#part7)
* [● Fermentation Addition Experiment](#part8)
* [Polyglutamic acid synthesis module](#part9)
* [● Gene cloning and plasmid construction](#part10)
* [● ygay-50trc-gdhA gene integration](#part11)
* [● Fermentation Additions Experiment](#part12)
* [Fermentation experiment of the complete pathway](#part13)
* [● Plasmid constrution](#part14)
* [● Additional experiment](#part15)

## Creatinine degradation module

### Gene cloning

#### 1.1 crnA

We revived Pseudomonas putida NBRC 14164 from our laboratory's bacterial culture collection and
extracted its complete genome using the TIANamp Bacteria DNA Kit (Cat.#DP302-02). Primers were
designed with KpnI and BamHI restriction enzyme sites (as shown in Figure 1) and amplified by
PCR (all primers, and formulations are provided in the supporting information).

![](https://static.igem.wiki/teams/4644/wiki/result111.png)

Figure1.crnA PCR results

#### 1.2 creJ

Since Arthrobacter nicotianae strain 23710 is not currently preserved in our laboratory, we
ordered the gene sequence from BGI Genomics. We amplified the sequence from the PMV plasmid with
the fused creJ gene obtained from the company. This sequence will be used for homologous
recombination with the pcs27 plasmid.

The amplification results are shown in Figure 2.

![](https://static.igem.wiki/teams/4644/wiki/result2.jpg)

Figure2.creJ(left)

### Co-expression plasmid construction

As part of the module validation, we wanted to investigate the performance data of the creatinine
degradation module. Therefore, we conducted separate fermentation and supplementation
experiments for this module. Considering the requirements for the expression levels of these two
enzymes, we selected the pCS27 plasmid with medium copy number and used the lac promoter to
express the desired proteins under IPTG induction.

To do this, we separately constructed two plasmids, pCS-lac-crnA and pCS-lac-creJ, with the
primers as mentioned above. Additionally, we amplified the plasmid backbone of pCS27 and the
homologous recombination fragment of the glutamate racemase (details can be found in the
complete pathway fermentation experiment). We first connected the three fragments using overlap
PCR and then performed homologous recombination with the plasmid backbone to obtain the required
plasmid for this module.

The plasmid map is shown in the figure.

![](https://static.igem.wiki/teams/4644/wiki/result2.png)

Figure3. pCS27-lac-crnA-creJ-racE

### Fermentation Additive Experiment

#### 3.1Amplification and Validation of Plasmids

We will amplify the constructed plasmid in E.coli DH5α through chemical transformation method.
The transformed cells will be spread onto solid LB agar plates supplemented with kanamycin for
antibiotic selection. Positive colonies will be selected (Figure 4) and subjected to colony PCR
screening (Figure 5) using primers overlapping the target region. The colonies carrying the
correct plasmid will be cultured in liquid LB medium supplemented with kanamycin at 37°C on a
shaker overnight. Plasmid DNA will be subsequently extracted. To ensure the correctness of the
plasmid sequence, it will be sent to a genetic company for sequencing, confirming its
accuracy.

![](https://static.igem.wiki/teams/4644/wiki/result4.jpg)

Figure4.Plasmid Transformation Results

![](https://static.igem.wiki/teams/4644/wiki/result5.jpg)

Figure5.Colony PCR Results

#### 3.2Additional experiments

**Experimental Method:**  The carrier bacterium to be used, Nissle 1917 (EcN), was pre-cultured
overnight at 37°C in LB medium without antibiotics. Plasmid transformation was carried out using
electroporation, and the transformed plasmids were selected on solid LB medium with kanamycin
resistance. Positive colonies were picked and inoculated into liquid LB medium with kanamycin
resistance, followed by overnight incubation at 37°C. Subsequently, they were inoculated into 50
mL of M9 medium with kanamycin resistance, induced with IPTG, and supplemented with the
substrate creatinine (1 mM). The cultures were then incubated on a shaker at 30°C for 48 hours.

![](https://static.igem.wiki/teams/4644/wiki/result6.jpg)

Figure6.Electroporation Results

At 12, 24, 36, and 48 hours of fermentation, samples were taken from the fermentation broth,
processed, and subjected to High-Performance Liquid Chromatography (HPLC) analysis to obtain the
fermentation data for this module.

![](https://static.igem.wiki/teams/4644/wiki/result7.jpg)

Figure7. The consumption of creatinine and the production of urea in the experimental group

![](https://static.igem.wiki/teams/4644/wiki/result8.jpg)

Figure8. The consumption of creatinine and the production of urea in
the
blank control group.

Compared with the blank control, our results show that the strain after our transformation can metabolize creatinine and transform it well, which symbolizes that the construction of our first module is successful, which is a very gratifying result. At the same time, through the investigation of the literature, we found that our results were close to the existing literature reports.Compared with the blank control, our results show that the strain after our transformation can metabolize creatinine and transform it well, which symbolizes that the construction of our first module is successful, which is a very gratifying result. At the same time, through the investigation of the literature, we found that our results were close to the existing literature reports.

## Urea degradation module

### Gene cloning

#### 1.1 UreABC

As part of the structural protein of urease, the gene cluster of urease has been widely
reported. We used the urease gene cluster from Providencia rettgeri strain JNB815. Since this
strain was not preserved in our laboratory, we purchased it from the China Center of Industrial
Culture Collection (CICC).

Similarly, we revived this strain and extracted its genome using the same method. We amplified
this sequence through PCR. Considering expression levels, we used our laboratory's high-copy
plasmid, pETlac, as the vector and performed homologous recombination with the gene clone.

![](https://static.igem.wiki/teams/4644/wiki/result9.png)

Figure9.ureABC

![](https://static.igem.wiki/teams/4644/wiki/result10.png)

Figure10.pETlac-ureABC

#### 1.2 ureEFGD

As an important auxiliary structure of urease, we also obtained it from the genome of Providencia
rettgeri strain JNB815 and recombined it into the pETlac plasmid using the same method.

![](https://static.igem.wiki/teams/4644/wiki/result11.png)

Figure11. ureEFGD

![](https://static.igem.wiki/teams/4644/wiki/result12.png)

Figure12.pETlac-ureEFGD

### Co-expression plasmid construction

Until now, there have been very few reports on the soluble expression of urease in Providencia
rettgeri strain JNB815. Using pETlac-ureABC and pETlac-ureEFGD as templates, we amplified the
two expression frames separately. After connecting them, we homologously recombined them with
the pETlac plasmid backbone to obtain this plasmid."

![](https://static.igem.wiki/teams/4644/wiki/result13.png)

Figure13.lac-ureABC-lac-ureEFGD

![](https://static.igem.wiki/teams/4644/wiki/result14.png)

Figure14.pETlac-ureABC-lac-ureEFGD

### Fermentation Addition Experiment

#### 3.1 Plasmid Amplification and Verification

We will amplify the constructed plasmid by chemical transformation in E. coli DH5α and then
spread it on ampicillin-resistant solid LB agar plates for resistance selection. Positive
colonies will be selected (Figure 15) and subjected to colony PCR screening (Figure 5, using
ureABC as PCR primers) to obtain strains carrying the correct plasmid. These strains will be
cultured in liquid LB medium supplemented with ampicillin resistance at 37°C with shaking
overnight. Subsequently, plasmids will be extracted to ensure the correctness of the plasmid
sequence. They will be sent to a gene company for sequencing to confirm their accuracy.

![](https://static.igem.wiki/teams/4644/wiki/result15.png)

Figure15.pETlac-ureABC-lac-ureEFGD Colony PCR Results（right）

#### 3.2 Soluble Expression of Urease

In E. coli expression systems, inclusion bodies can form when expressing foreign proteins due to
the lack of modification systems. Currently, the mainstream approach in the scientific community
to increase solubility is to purify and refold the expressed proteins or use molecular
chaperones or solubilization tags. Therefore, BUCT has attempted four molecular chaperones to
enhance solubility: GroEL/GroES, DnaK/DnaJ, IbpAB, and sigma32. The plasmid maps for each of
these chaperones are shown in the figure below.

![](https://static.igem.wiki/teams/4644/wiki/bbbbbbb.png)

**Solubility Verification:**

The gene segments constructed as described above were subjected to homologous recombination with
the pET-duet plasmid using the same method. E. coli BL21 cells, which had been pre-cultured and
stored, were revived. The plasmids were transformed into the host cells through electroporation.
Four different molecular chaperones were separately introduced into the host cells, and a
control group with an empty vector and another control group with no molecular chaperones were
included, resulting in a total of six groups.After selection for antibiotic resistance, the
cells were initially inoculated into LB medium containing both ampicillin and kanamycin
antibiotics. Subsequently, they were transferred to 50 ml of LB medium containing both
antibiotics in shake flasks and cultured at 37°C until the OD reached the desired range
(0.2-0.6). The cultures were induced with 0.5 mM IPTG and further incubated for 12-16 hours.The
fermentation broth was then collected and subjected to repeated sonication for cell disruption.
Each group was divided into two fractions: the supernatant and the precipitate. Finally, the gel
electrophoresis results for this protein were obtained using SDS-PAGE.

![](https://static.igem.wiki/teams/4644/wiki/result20.png)

Figure20.Urease SDS-PAGE-(1)

![](https://static.igem.wiki/teams/4644/wiki/result21.png)

Figure20.Urease SDS-PAGE-(2)

After comparison, it is easy to notice that the effect of GroEL/GroES is the most excellent.

#### 3.4 Additional experiments

**Experimental Method:**

The carrier strain, Nissle 1917 (EcN), to be used in the experiment is pre-cultured overnight at
37°C in LB medium without antibiotics. The co-expression plasmid and chaperone plasmid are
transformed into the carrier strain using electroporation. The transformed cells are then
screened on solid LB medium containing both ampicillin and kanamycin as dual resistance markers.
Positive colonies are selected and inoculated in liquid LB medium containing ampicillin and
kanamycin at 37°C overnight. The cells are subsequently inoculated into 50 ml of M9 medium
containing ampicillin and kanamycin, induced with IPTG (Isopropyl β-D-1-thiogalactopyranoside),
and supplemented with urea (10g/L). The cultures are incubated in a shaker at 30°C for 48 hours.

Samples of the fermentation broth are taken at 12, 24, 36, and 48 hours of fermentation. The
samples are processed and analyzed using High-Performance Liquid Chromatography (HPLC) to obtain
fermentation data for this module. Due to the inherent metabolic activities of Escherichia coli,
it is difficult to measure the exact amount of ammonia produced. However, fortunately, E. coli
itself cannot directly metabolize urea. Therefore, the decrease in urea concentration can
directly reflect the performance of this module.

![](https://static.igem.wiki/teams/4644/wiki/result22.jpg)

Figure21.Urea consumption in the experimental group

![](https://static.igem.wiki/teams/4644/wiki/result23.jpg)

Figure22. Urea consumption in the control group

In fact, although we have made a lot of efforts, including literature research and group discussion, we still haven't got an absolutely effective solution to the problem of urease soluble expression, which is undoubtedly regrettable, but we also know that the process of scientific research is full of challenges, so we will continue to explore this problem and strive to solve this problem!

## Polyglutamic acid synthesis module

### Gene cloning and plasmid construction

#### 1.1 racE

Because the synthesis of polyglutamic acid requires equal amounts of L-glutamic acid and
D-glutamic acid, the addition of glutamate racemase can increase the yield of polyglutamic acid.
Therefore, we identified the glutamate racemase gene racE in Bacillus amyloliquefaciens LL3 and
directly ordered it from a genetic company. We amplified the gene fragment for homologous
recombination directly from the obtained PMV plasmid containing this gene. This gene fragment
was then homologously recombined with the pcs27 plasmid backbone to obtain pcs-lac-racE.

#### 1.2 pgsBCAE

The pgs promoter from Bacillus subtilis strain A-5 plays a crucial role in the synthesis of
polyglutamic acid. It utilizes racemic glutamic acid as a raw material to synthesize
polyglutamic acid and then expels the product extracellularly. We obtained it through PCR from
the genome of Bacillus subtilis strain A-5 and recombined it with the pETlac plasmid
backbone.

![](https://static.igem.wiki/teams/4644/wiki/result24.png)

Figure23 pETlac-pgsBCAE

### ygay-50trc-gdhA gene integration

Because this module requires glutamic acid as a precursor, and in the metabolic system of E.
coli, the gdh pathway is one of the major pathways for glutamic acid production. Therefore, we
identified the gene encoding glutamate dehydrogenase, gdhA, in E. coli. We replaced the
constitutive promoter -50trc. Additionally, to reduce the burden on the bacterial cells, we used
CRISPR-Cas9 technology to target the ygaY locus in the genome for integration. After integration
was completed, we induced the elimination of the PT plasmid using L-arabinose and cultured the
cells at 42°C for 12 hours to eliminate the pCas9 plasmid.

![](https://static.igem.wiki/teams/4644/wiki/result25.png)

Figure24.ygay-50trc-gdhA

![](https://static.igem.wiki/teams/4644/wiki/result26.jpg)

Figure25. Integration Strain Plate Streaking Chart

### Fermentation Additions Experiment

Because the gene-integrated vector strain is still under preparation prior to this fermentation
experiment, the wild strain is still being used for fermentation.

**Method:**

The carrier strain to be used, Nissle 1917 (EcN), was pre-cultured overnight at 37°C in LB medium
without antibiotics. Both plasmids mentioned above were co-transformed using electroporation,
and positive colonies were selected on solid LB medium with double resistance to ampicillin and
kanamycin. The selected colonies were then inoculated into liquid LB medium with double
resistance to ampicillin and kanamycin and cultured overnight at 37°C. Subsequently, they were
inoculated into 50 ml of M9 medium with double resistance to ampicillin and kanamycin, induced
with IPTG, and supplemented with the substrate glutamic acid (5g/L). The cultures were shaken at
30°C for 48 hours.

At the 12th, 24th, 36th, and 48th hours of fermentation, samples of the fermentation broth were
taken respectively. After processing the samples, they were subjected to High-Performance Liquid
Chromatography (HPLC) analysis to obtain fermentation data for this module.

![](https://static.igem.wiki/teams/4644/wiki/result27.jpg)

Figure26. The experimental group's production of polyglutamic acid

![](https://static.igem.wiki/teams/4644/wiki/result28.jpg)

Figure27. The experimental group's production of polyglutamic acid

The experimental results show that our construction of the pathway of γ-PGA is quite successful. The modified E. coli can effectively convert glutamate into γ-PGA. Although the yield cannot reach the highest yield in the world at present, this may be related to the use of different carrier bacteria, but this result still shows that our construction of polyglutamic acid synthesis module is successful.

## Fermentation experiment of the complete pathway

### Plasmid constrution

In order to construct a complete metabolic pathway, we will create two co-expression
plasmids.

#### 1.1 pCS27-lac-crnA-creJ-racE

Considering the requirements for expression levels, excessive expression in the front part of the
pathway can lead to the accumulation of intermediates such as ammonia, which has a negative
impact on the strain. In addition, based on reports regarding the activity of various enzymes,
we have made certain adjustments to the gene positions.

The construction method of this plasmid has been described in detail in the creatinine
degradation module.

![](https://static.igem.wiki/teams/4644/wiki/result29.png)

Figure28.pCS27-lac-crnA-creJ-racE

#### 1.2 pETlac-ureABC-plpp1.2-ureEFGD-lac-pgsBCA

After conducting experiments in the urea degradation module, we found that even with the
assistance of molecular chaperones, the activity of urease was relatively weak compared to other
enzymes in the pathway. Recognizing the significance of accessory proteins in enhancing its
enzymatic activity, we decided to replace the constitutive promoter with a higher strength one
in an attempt to increase urease activity. To do this, we first used the pZe12 plasmid for
homologous recombination to obtain pZe-plpp1.2-ureEFGD. Then, we amplified the entire expression
cassette through PCR and, using the same method, recombined the resulting lac-ureABC and
lac-pgsBCAE with the pETlac plasmid backbone after overlap connection.

![](https://static.igem.wiki/teams/4644/wiki/result30.png)

Figure29. pETlac-ureABC-plpp1.2-ureEFGD-lac-pgsBCAE

### Additional experiment

After successfully constructing the plasmids and confirming their integrity through company
sequencing, our operational procedure was similar to the previous one, with the exception that
we used an engineered integrative strain as the host. The host strain we used was Nissle 1917
(EcN), which had been integrated with ygay-50trc-gdhA. This strain was pre-cultured overnight at
37°C in LB medium without antibiotics. Subsequently, we performed co-transformation of the two
plasmids and the molecular chaperone using electroporation. Selection was carried out on solid
LB medium containing both ampicillin and kanamycin, and positive colonies were picked and
inoculated into liquid LB medium containing both antibiotics. The culture was incubated at 37°C
overnight.

The next step involved inoculating the strain into 50 mL of M9 medium without a nitrogen source
but with dual resistance to ampicillin and kanamycin. After induction with IPTG, we added
substrates creatinine (1 mM) and urea (10 g/L) and cultured the cells on a shaker at 30°C for 48
hours.

At 12, 24, 36, and 48 hours of fermentation, samples were taken from the culture. These samples
were processed and subjected to High-Performance Liquid Chromatography (HPLC) analysis, yielding
fermentation data for this module.

![](https://static.igem.wiki/teams/4644/wiki/result31.jpg)

Figure30. Experimental Group: Urea is represented by its consumption,
while all other substances are represented by their production

![](https://static.igem.wiki/teams/4644/wiki/result32.jpg)

Figure31. Control group

The construction of the complete pathway and the results of the fermentation experiment are quite exciting, as it indicates that we have successfully constructed this pathway. Although our production is relatively low, this may be due to the poor actual effect of urease, it still marks a milestone step for BUCT in solving the pain of CKD patients!

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