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.

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.

But the performence in microaerobic situation is also inhibited.

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).

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).

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.

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

After collating and analyzing the fluorescence intensity data, it can be seen obviously that the improved promoter (Pvgb-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.

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:

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

Divide experimental groupF3，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.

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.