iGEM 2022 — Team njtech-china

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Notebooks

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**Engineering Success**

**Construction of the pMTL-ΔperR plasmid**

**[DESIGN]**

PerR or its homologous protein is the transcriptional repressor involved in the H2O2 stress response in Firmicutes, which deletion is reported to enable a rapid induction of genes required to protect cells from a H2O2 stresses.

The role of the PerR repressor has been studied in *C. acetobutylicum* where it is proposed that this regulator acts as a switch to O2 tolerance, and the genes encoding the components of the alternative detoxification system were found to be PerR regulated. While a wild-type *C. acetobutylicum* strain loses viability after exposure to H2O2 or molecular oxygen, the inactivation of the PerRCA repressor leads to a better resistance to H2O2 as observed in other Firmicutes.

We want to analyze the growth of *C. tyrobutyricum* after knocking out *perR* in its genome, so we constructed this plasmid for the deletion of *perR*.

![](https://static.igem.wiki/teams/4119/wiki/eng11/1.jpg)

**Fig-1 The expression vector of the pMTL-ΔperR plasmid**

**[BUILD]**

The upper and lower homology arms (HR-UP, HR-DOWN) were constructed at about 500 bp upstream and downstream of the PerR sequence of the *C. tyrobutyricum* genome, plus a lactose-inducing promoter and a spacer sequence derived from the PerR , generating the CRISPR array . Using the plasmid pMTL82151 from *C. tyrobutyricum* as a template, a linear vector was obtained by EcoRI and KPnI double digestion. The CRISPR array and linearized vector after double digestion were ligated using Gibson's assembly method. Colony PCR is performed on the transformed colonies. The positive colonies with correct colony PCR were transferred and the plasmid was picked, and the recombinant plasmid was obtained after sequencing verification: pMTL-perR-HR.

**[TEST]**

**The growth profiles of the plasmid knockout strain under different contents of oxygen**

After the experimental strain was activated by TGA plate medium, the single colony was selected and incubated at 37 °C in TGA liquid medium until the OD600 was about 1.0 as fermentation seed solution, and transferred to a 50 mL volume centrifuge tube containing 15 mL TGA medium according to the 5% volume specific inoculation amount, and placed in 37 °C 0r, 50 r, 100 r fermentation for 80 h respectively for product detection.

**Colony morphology observation**

After the experimental strain was activated by TGA plate medium, the single colony was selected and incubated at 37 °C in TGA liquid medium until the OD600 was about 1.0 as seed solution, and transferred to a 50 mL volume centrifuge tube containing 15 mL TGA medium according to the 5% volume specific inoculation amount, and placed in 37 °C 0r, 50 r, 100 r cultivate for 80 h respectively for growth detection.

**Results**

As can be seen from the sequencing results, the perR fragment has been knocked out.

![](https://static.igem.wiki/teams/4119/wiki/engineer1/3.png)

**Fig 2. Sequencing verification of obtained mutant. The red part represents the absence of the sequence.**

At different rotational speeds, the growth of the plasmid knockout strain was better than that of the control strain.

![](https://static.igem.wiki/teams/4119/wiki/engineer1/4.png)

**Fig 3. The growth profiles of Ct (ΔperR) and Ct (Control) under different contents of oxygen**

![](https://static.igem.wiki/teams/4119/wiki/engineer1/5.png)

**Fig 4. Physiological characteristics of Ct (ΔperR) and Ct (Control) in the presence of oxygen. (A) The intracellular ROS levels in Ct (ΔperR) and Ct (Control) in the presence of oxygen. ROS levels were measured in the logarithmic prophase. (B) The ratio of NADH/ NAD+ in Ct (ΔperR) and Ct (Control) in the presence of oxygen. Phase I, II and III refer to the early-, mid- and late- logarithmic phase, respectively.**

![](https://static.igem.wiki/teams/4119/wiki/engineer1/6.png)

**Fig 5. The survival rate of Ct (ΔperR) and Ct (Control) to oxidative stress.**

**[Learn]**

In the experiments, we found that the knockout plasmid was still present in *C. tyrobutyricum* after completing the knockout of perR , which somewhat hindered the introduction of the subsequent plasmids. In reviewing the literature we learned that, tetR is a repressant. In the absence of tetracycline, tetR binds to the tetracycline operon to represses the transcription of the tetracycline overflow pump gene. When tetracycline enters the cell, tetracycline binds to tetR and changes its conformation, then tetracycline operon is not hindered and can express the CRISPR array behind it normally，thus knocking itself out. Therefore, we hope to eliminate the imported plasmids by adding the tetR fragment to the plasmid and then by tetracycline induction at a later stage.

**Construction of the Plac->ΔperR -> self - target plasmid**

**[DESIGN]**

As the previously constructed pMTL-ΔperR plasmid has been introduced into *C. tyrobutyricum*, introducing other plasmids like pMTL-Pvgb-perR could be difficult, and the growth of the bacteria would be impacted, so we expected that the plasmid using for knocking out perR could be degraded after completing its task, and that leads us to the design of the Plac->ΔperR->self-target plasmid.

The tetR gene encodes the tetracycline repressor protein(TetR). And in the absence of tetracycline, it binds to the tetracycline operon(tet operon, tetO) to inhibit the transcription of the plasmid self targeting CRISPR array. When tetracycline is added, TetR binds to tetracycline instead, and the transcription of downstream CRISPR array would be carried out and the plasmid will be degraded.

![](https://static.igem.wiki/teams/4119/wiki/eng11/2.jpg)
![](https://static.igem.wiki/teams/4119/wiki/eng11/3.jpg)

**Fig-6 The expression vector of the Plac->ΔperR -> self - target plasmid and its mechanism**

**[BUILD]**

Use the previously constructed pMTL-ΔperR as a template to obtain pMTL-ΔperR linearized vector by PCR amplification, and extract *C. tyrobutyricum* genome as a template to amplify the tetR fragment with homologous arms. Then the tetR fragment and the pMTL-ΔperR linearized vector were connected using Gibson-assembled method by homologous recombination. Colony PCR was performed on the transformed colonies, and the positive colonies with correct agarose gel band were transferred and cultured. The plasmids were extracted for sequencing verification, and correct plasmids would be obtained.

**[TEST]**

**Characterization experiment**

Our plasmid constructed contained methsulfamycin resistance gene for inducing knockdown of the perR gene with 40mmol/L lactose in RCM medium at 3μl/mL Tm and 2.5ul/mL D-cycloserine, after induction of 8-10 generations, the coating plate is diluted and the single colony is subjected to colony PCR to verify that perR is successfully knocked out. After successful knockout of the perR on the genome, followed by a certain concentration of tetracycline addition in non-resistant RCM medium. After induction 8 – 10 generations, we obtained single colonies by dilution coated plate for colony PCR validation.

![](https://static.igem.wiki/teams/4119/wiki/engineer1/9.png)

**Fig 2. Plasmid-self-target bacteria colonies PCR results**

The results show that the target plasmid colonies PCR results appear band to eliminate the plasmid bacteria no band, at the same time, eliminate the target plasmid bacteria cannot survive in the medium containing resistance, prove that the target bacteria has completed plasmid elimination.

We hope to induce the elimination of plasmids by tetracycline after lactose-induced knockout of the gene of interest, and at present, we have only achieved lactose-induced knockout of the gene of interest with success. In addition, we designed plasmids solely to eliminate plasmids to verify whether the method of eliminating plasmids using tetr is feasible in C. tyrobutyricum. We introduced a plasmid carrying the tetR gene in C. tyrobutyricum and successfully eliminated it by tetracycline induction, demonstrating that this method is feasible. The work of knocking out the gene of interest and then eliminating the plasmid for time reasons is not completed.

**[Learn]**

In the early experiments using lactose inducible promoter to regulate the expression of genes, but in the actual process, especially perR gene knockout process, we found that although has induced many generations, but always cannot induce gene expression, in the literature we learned that because we choose RCM medium contains a high proportion of glucose, and glucose and lactose will produce fusion phenomenon so that lactose can not play a good role on our gene induction. This is a large part of the slowness of our experiments. We then wished to perform induced knockdown by the xylose promoter that does not undergo a fusion phenomenon with glucose.

**Construction of the pMTL-P*thl*-perR plasmid**

**[DESIGN]**

The growth data of *C. tyrobutyricum* when perR gene is not expressed can be obtained by constructing plasmid with knock-out perR gene and introducing it into *C. tyrobutyricum*. We also hope to find out whether excessive expression of perR gene will inhibit the growth of flora compared with wild *C. tyrobutyricum*, so we choose to use constitutive promoter P*thl* to over-express perR gene and observe the growth of flora after expression.

![](https://static.igem.wiki/teams/4119/wiki/engineer2/1.png)

**[BUILD]**

The main chain pMTL82151 of plasmid was digested by BamHI enzyme, and the linear vector was obtained. Using *C. tyrobutyricum* genome as template, PerR gene fragment was amplified by PCR.

Gibson assembly method was used to link PerR gene fragment with linearized pMTL82151 vector. The transformed colonies were detected by colony PCR. The correct (positive) colony was cultured for plasmid extraction, and verified by nucleotide sequencing to obtain the recombinant plasmid: pMTL-P*thl*-perR.

**[TEST]**

**The growth profiles of the Engineered strains under different contents of oxygen**

After the experimental strain was activated by TGA plate medium, the single colony was selected and incubated at 37 °C in TGA liquid medium until the OD600 was about 1.0 as seed solution, and transferred to a 50 mL volume centrifuge tube containing 15 mL TGA medium according to the 5% volume specific inoculation amount, and placed in 37 °C 0r, 50 r, 100 r cultivate for 80 h respectively for growth detection.

![](https://static.igem.wiki/teams/4119/wiki/engineer2/2.png)

**Fig 4- Physiological characteristics of Ct (Pthl-perR) and Ct (Control) in the presence of oxygen.**

**[LEARN]**

We found that by knockout of perR gene, *C. tyrobutyricum* growth is inhibited under anaerobic conditions, and the overexpression of perR gene *C. tyrobutyricum* is inhibited in growth under aerobic conditions, so we wanted to regulate perR gene expression by the oxygen-inducible promoter, and then *C. tyrobutyricum* has higher oxygen resistance.

**Construction of the pMTL-P*vgb*-Bs2 plasmid**

**[DESIGN]**

In this part of our project, the key promoter *vgb* was a microaerobic induced promoter of *Vitreoscilla* hemoglobin gene. Considering the gene compatibility difference between different host bacteria, we designed the pMTL-P*vgb*-bs2 plasmid to determine whether the promoter *vgb* could work normally in *C. tyrobutyricum* by detecting the fluorescent expression intensity of fluorescent protein Bs2.

![](https://static.igem.wiki/teams/4119/wiki/engineer2/3.png)

**[BUILD]**

The linear vector was obtained by single digestion of plasmid backbone pMTL82151 using Bam HI enzyme. The recombinant plasmid pMTL-P*thl*-Bs2 offered by Dr. Zhengming Zhu’s research group was used as the template to generate the Bs2 fragment. The Bs2 fragment was then used as template and promoter vgb sequence was constructed upstream by PCR technique to obtain fragment P*vgb*-Bs2, meanwhile a pair of homologous arms was added both side for recombination.

The P*vgb*-Bs2 fragment and the pMTL82151 linearized vector were linked by Gibson assembly method, and colony PCR was performed on the transformed colonies. The correct(positive) colonies were cultured for plasmid extraction, and those plasmid were verified by nucleotide sequencing to obtain the recombinant plasmid: pMTL-P*vgb*-Bs2.

**[TEST]**

**Fluorescence analysis

Due to the tight time we chose to verify the promoter in *E. coli* first.The bacteria introduced with pMTL-P*vgb*-Bs2, pMTL-P*thl*-Bs2 and pMTL82151 plasmids were precultured, inoculated into LB culture medium with different dissolved oxygen, and cultured to OD600 ~0.7 and ~1.2, respectively. Two milliliters of bacterial culture solution were taken, centrifuged at 12,000 rpm for 2min, washed with 700ul 1xPBS buffer for 2 times. After resuspending the bacterial pellet using another 700ul 1x PBS buffer, 200 μl of that solution were pipetted into a 96-well plate for microplate reader analysis, in which the obtained data was analyzed.

**Result**

The test group: the fluorescence intensity is relatively high

The control group: the fluorescence intensity is lower compared to the test group

![](https://static.igem.wiki/teams/4119/wiki/engineer2/5.jpg)

**[learn]**

We wanted to express Bs2 fluorescent protein by using the oxygen- inducible promoter *vgb*, and used the expression of fluorescence intensity to verify its situation under oxygen regulation in *C. tyrobutyricum*. Due to limited time, we did its expression in *E. coli*, and the data showed that its fluorescence intensity in microoxygen is much greater than that in high dissolved oxygen amount. Therefore, in subsequent experiments, we replaced the strong promoter *thl* with this promoter

**Construction of the pMTL-Pvgb-perR plasmid**

**[DESIGN]**

P*vgb* is an oxygen-dissolving regulated promoter from *Vitreoscilla*. We have done its expression effect in *C. tyrobutyricum* before, which proves that it can express bs2 fluorescent protein in different amounts under different oxygen concentrations of *C. tyrobutyricum*. On the premise that the expression of perR gene can affect the growth of *C. tyrobutyricum*, We hope that pvgb promoter can be connected with perR gene by Gibson assembly method, and perR gene can be expressed in different degrees by pvgb promoter and the conditions of controlling different oxygen concentrations, so that *C. tyrobutyricum* has higher oxygen adaptability.

![](https://static.igem.wiki/teams/4119/wiki/engineer2/6.png)

**[BUILD]**

Using the recombinant plasmid pMTL-P*thl*-Bs2 provided by Dr. Zhu Zhengming's research team as a template, the pMTL-P*vgb* linearized vector was amplified. With pMTL-P*thl*-perR as template, the PerR gene fragment was amplified. Gibson assembly method was used to link PerR gene fragment with pMTL-P*vgb* linearization vector. And the transformed colonies were detected by colony PCR. The correct (positive) colony was cultured for plasmid extraction, and verified by nucleotide sequencing to obtain the recombinant plasmid: pMTL-p*vgb*-perR.

**[TEST]**

**The growth profiles of the Engineered strains under different contents of oxygen**

After the experimental strain was activated by TGA plate medium, the single colony was selected and incubated at 37 °C in TGA liquid medium until the OD600 was about 1.0 as seed solution, and transferred to a 50 mL volume centrifuge tube containing 15 mL TGA medium according to the 5% volume specific inoculation amount, and placed in 37 °C 0r, 50 r, 100 r cultivate for 80 h respectively for growth detection.

![](https://static.igem.wiki/teams/4119/wiki/engineer2/7.jpg)

**[Expect]**

In subsequent experiments, we will further test the effect of dynamic regulation of perR by P*vgb* on the growth of *C. casitirate*, and we expect to achieve better growth than wild bacteria in both microoxygen and aerobic conditions.**

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Experiments

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Results

**Oxygen-inhibitory modification of the constitutive promoter, PthlA**

**Strain construction**

Successful mapping of the colony pcr of the bacteria imported into the modified pMTL82151-PthlAF3-bs2, and the pMTL82151-PthlAF7-bs2 plasmid.
The plasmid pMTL82151-PthlA-bs2 was extracted using Vazyme’s FastPure Plasmid Mini Kit. Then，using pMTL82151-PthlA-bs2 as templates, carry out the PCR respectively with the *F3FNRbindingsite-F, F7 FNR bindingsite-F, and (check)FNR binding site-R*, obtain megaprimers of 908bp and 912bp.

Using pMTL82151-PthlA-bs2 as templates again, we obtained two plasmids of pMTL82151-PthlAF3-bs2 and pMTL82151-PthlAF7-bs2 with two large recombinant plasmids into E. coli JM109 competent cells. Spread the culture solutions on the agar plates. Subject the transformed colonies for colony PCR, using *F7 check FNR binding site-F, F3 check FNR binding site-F, and （check）FNR binding site-R*. Obtain fragments of 936bp and 940bp.Site-directed mutations were successfully proved.

Using pMTL-PthlAF7-bs2 as template, perform the PCR respectively with the Gibson Vector-F and Gibson Vector-R, obtaining linearized vector pMTL-PthlAF7-bs2. Using the genome of E. coli DH5αas the template and Gibson Fragment /genome FNR-F and Gibson Fragment /genome FNR-R as the primers, obtain fnr fragment. The genome of *Clostridium tyrobutyricum* were used as template, and Gibson Fragment/genome T7-cat1-F and Gibson Fragment/genome T7-cat1-R as primers，obtain fragment T7-cat1. The plasmid pMTL-PthlAF7-bs2-T7-cat1-FNR was assembled by Gibson using pMTL-PthlAF7-bs2 as the vector, fnr and T7-cat1 as fragments. Then transform the recombinant plasmid into E. coli JM109 competent cells, colony PCR.

**Fluorescence performance**

Divide experimental group F3,F7 and the positive control(pMTL82151-PthlA-bs2) into aerobic and Microaerobic groups. Perform fluorescence assays when the experimental groups, the F3,F7 and the positive controls(pMTL82151-PthlA-bs2) were all grown to the appropriate OD600(optical density at 600nm).

Conclusion: After modification, it’s excepted that for group F3 under microaerobic condition, other experimental groups had lower expression levels than the positive control group. The fluorescence intensity shows that the promoter thlAF7 has a trends in aerobic inhibition. But the performance in microaerobic situation is also inhibited.

The new promoter thlAF3 has a satisfied performance. It inhibits the expression of its downstream genes under aerobic conditions while strengthens that in microaerobic condition as we wish.

![](https://static.igem.wiki/teams/4119/wiki/result/1.jpg)

**Fig1.Fluorescence intensity of F7 group and positive control group under different oxygen conditions**

But the performence in microaerobic situation is also inhibited.

![](https://static.igem.wiki/teams/4119/wiki/result/2.jpg)

**Fig2.Fluorescence intensity of F3 group and positive control group under different oxygen conditions**

**Doubling promoter vgb to enhance the expression level of the promoter.**

Conclusion:One pairs of verification primers (pMTL-jp-F; pMTL-jp-R) were used to verify the modified plasmid by PCR. The correct bands for PCR using pMTL-jp-F; pMTL-jp-R primers were 484 bp. The bands of strain pMTL-P2vgb-bs2 were correct(484bp).

![](https://static.igem.wiki/teams/4119/wiki/eng11/8.png)

**Fig3. Verification gel diagram of PCR with pMTL-jp-F and pMTL-jp-R primers**

M:DL1000 Plus DNA Marker

1-5:pMTL-P2vgb-bs2

Conclusion:One pairs of verification primers (pMTL-jp-F; pMTL-jp-R) were used to verify the modified plasmid by PCR. The correct bands for PCR using pMTL-jp-F; pMTL-jp-R primers were 484 bp.The band of the pMTL-P2vgb-bs2-1 and pMTL-P2vgb-bs2-3 strain was correct (484bp).

**Characterizaion the Pvgb and P2vgb Promoters**

![](https://static.igem.wiki/teams/4119/wiki/result/4.jpg)

**Fig4. Fluorescence intensity of experimental group and control group under different oxygen conditions**

Conclusion:

1. The inhibition effect of the improved promoter(P2vgb) was stronger than that of the control group(Pvgb) under aerobic conditions.

2. Under the induction of microaerobic conditions, the improved promoter(P2vgb) behaves similarly to the control group(Pvgb) under aerobic conditions.

3. This suggests that tandem promoters improve protein expression.

**Inserting exogenous 5'-UTR sequences downstream of the promoter vgb to enhance the expression level of the promoter.**

By controlling aerobic and micro-aerobic culture conditions, we incubated E. coli bearing recombinant plasmid P*vgb-5’-UTR-bs2* together with P*vgb-bs2* as control, under those two conditions respectively, and sampled the culture solutions after logarithmic stages to detect fluorescence intensity after relevant treatment.

![](https://static.igem.wiki/teams/4119/wiki/result/5.jpg)

**Fig5.Expression effect of Pvgb-5’UTR-Bs2 at different oxygen concentrations**

After collating and analyzing the fluorescence intensity data, it can be seen obviously that the improved promoter (P*vgb-5’-UTR*) under aerobic conditions behaves similarly to the control
group (Pvgb), while under the induction of microaerobic conditions, the expression effect of the improved promoter is 1.97-folds of the control group, which indicates that the promoter engineering strategy of inserting exogenous 5’-untranslated region(5'-UTR) does improve the expression of downstream proteins.

**Inserting another FNR binding site at 8 bp upstream the native FNR regulatory region of promoter vgb to enhance the inhibitory effect of oxygen on the promoter.**

**Strain construction**

Successful mapping of the colony pcr of the bacteria imported into the modified pMTL82151-PvgbF3-Bs2, and the pMTL82151-PvgbF7-Bs2 plasmid.
The plasmid pMTL82151-Pvgb-Bs2 was extracted using Vazyme’s FastPure Plasmid Mini Kit.
Then,Using pMTL82151-Pvgb-Bs2 as templates, perform the PCR respectively with the VFBSF3-F, VFBSF7-F, and (check) -VFBS-R,obtain megaprimers of 668bp and 672bp.Using pMTL82151-Pvgb-Bs2 as templates again,obtain two plasmids of pMTL82151-PvgbF3-Bs2 and pMTL82151-PvgbF7-Bs2 with two large recombinant plasmids into JM109 competent cells.Spread the culture solutions on the agar plates.Subject the transformed colonies for colony PCR,using check-VFBSF3-F, check-VFBSF7-F, and (check) -VFBS-R.Obtain fragments of 639bp and 643bp.Site-directed mutations were successfully proved.The gel figure is as follows:

![](https://static.igem.wiki/teams/4119/wiki/result/6.png)

**Fig6.Rubber plot of the bacterial colony pcr**

Conclusion:pMTL82151-PvgbF3-Bs2 and pMTL82151-PvgbF3-Bs2 plasmid has been imported into JM109.

**Fluorescence performance**

Divide experimental group*F3，F7* and the positive control(pMTL82151-Pvgb-Bs2)into aerobic and microaerobic groups. Perform fluorescence assays when the experimental groups, the *F3, F7* and the positive controls (pMTL82151-Pvgb-Bs2) were all grown to the appropriate OD600.

![](https://static.igem.wiki/teams/4119/wiki/result/7.jpg)

**Fig7. Fluorescence expression of experimental groupF3,F7 and the positive control at different oxygen concentrations**

**Conclusion:** After modification, both experimental groups had lower expression levels than the positive control group, and we found that the pMTL82151-PvgbF3-Bs2 experimental group did not achieve the purpose of the transformation. However,the pMTL82151-PvgbF7-Bs2 experimental group showed aerobic inhibition and microoxide expression characteristics.

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