We aimed to insert an FNR binding site 7 bp away in front of the -35 region of the thlA promoter, which we wished to be able to insert by PCR amplification. Therefore, we designed primers containing FNR binding sites capable of specifically binding to the thlA promoter, then amplified a fragment by using another primer bound to plasmid pMTL82151. Then amplified the entire plasmid as a megaprimer, and carried out transformation of Escherichia coli. The fluorescence intensity shows that the engineered promoter has a trends in aerobic inhibition. However the performence of the promoter in microaerobic situantion was not as well as the origin promoter.

The fluorescence intensity shows that the remodeled promoter has a trends in aerobic inhibition. But the performence in microaerobic situation is also inhibited.

Because the fluorescence results of the strain containing pMTL-PthlAF7-bs2 under microaerobic conditions were not very good, we decided to shorten the distance between the FNR binding site and the -35 region to 3 bp. We similarly used PCR to insert an FNR binding site at the position of the first 3 BP of the -35 region. After the product of the first PCR had been obtained, we use it as a megaprimer to amplify the entire plasmid, then transform the plasmid into Escherichia coli. The fluorescence intensity shows us that the latest engineered promoter has an significant improve in aerobic inhibition. Moreover the new promoter also has a excellent improve in microaerobic situantion than the origin promoter.

The new promoter thlAF3 has a satisfatory performence. It is inhibited in aerobic condition while strengthed in microaerobic condition.

Trough literature research and preliminary experiments, we found that the expression of the vgb promoter was not ideal, so we tried to enhance its ability to express proteins by doubling promoter vgb.

We decided to insert a repeated P_vgb fragment into the upstream of P_vgb , hoping that the expression of Bs2 fluorescent protein could be improved.

We have converted the constructed and sequence analysed recombinant plasmid into E. coli CA434, but time remaining for the competition did not allow us to continue to transfer the recombined plasmid into C. tyrobutyricum by conjugation, so the detection of fluorescence intensity data were carried out in E. coli to verify the effectiveness of the improvements for the vgb promoter.

By controlling aerobic and micro-aerobic culture conditions, we incubated E. coli bearing recombinant plasmid pMTL- P_2vgb-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.

After collating and analyzing the fluorescence intensity data (Figure 1), it can be seen obviously that the inhibition effect of the improved promoter(P_2vgb) was stronger than that of the control group(P_vgb) under aerobic conditions. Under the induction of microaerobic conditions, the improved promoter(P_2vgb) behaves similarly to the control group(P_vgb) under aerobic conditions. This suggests that tandem promoters improve protein expression.

Trough literature research and preliminary experiments, we found that the expression of the vgb promoter was not ideal, so we tried to enhance its ability to express proteins by the insert of a fragment of exogenous 5’-untranslated region(5'-UTR).

In iGEM's official part registry catalog, we retrieved a translation enhanceing 5-UTR fragment(Part:BBa_K1758100) designed by team Bielefeld-CeBiTec in 2015, and we decided to insert this fragment downstream of the vgb promoter in the pre-constructed plasmid pMTL-vgb-bs2, hoping that the expression of the Bs2 fluorescent protein would improve. This sequence contains a 5'-UTR and a strong ribosomal binding site(RBS) from bacteriophage T7. This sequence could greatly enhance translation of a following gene. The enhancing effect relies on the regulation of mRNA binding to and release of the ribosome S30 subunit.

We have converted the constructed and sequence analysed recombinant plasmid into E. coli CA434, but time remaining for the competition did not allow us to continue to transfer the recombined plasmid into C. tyrobutyricum by conjugation, so the detection of fluorescence intensity data were carried out in E. coli to verify the effectiveness of the improvements for the vgb promoter.

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 (Figure 1), 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.

By consulting the literature and consulting the authors, we learned that the distance between the FNR binding site and the -35 region of the promoter has a large impact on promoter transcriptional regulation.

To change the distance of FNR binding site from the -35 region, we used site-directed mutagenesis. Large primer mutations technique was used to separate the FNR binding site from the -35 region by, changing the distance between the FNR binding site to 3bp and 7bp from -35 region, while changing the sequence from the second half of the FNR binding site and the -35 region to E. coli adaptation, reducing the interval between-35 and-10 region to 17bp.

The plasmid pMTL-P_vgb-bs2 was extracted from recombined E. coli CA434 pMTL-P_vgb-bs2 constructed previously.

In this experiment, the megaprimers were obtained by PCR technique using the plasmid pMTL-P_vgb-bs2 as the template, and then the mutant plasmid was obtained by PCR using those megaprimers to amplify the plasmid.

By analyzing the fluorescence intensity data, it can be found that the increase in the distance between the FNR binding site and the -35 region of the promoter could result in a certain decrease in its expression effect.

Under aerobic and microaerobic conditions, the modified promoter (P_vgb-F7) was separately 0.267 and 0.422 folds of the control group (Pvgb). The modified promoter still has the regulatory effect brought by FNR and its based oxygen-related biosensor system, which induction ratio increased to 6.28, compared with 3.97 of Pvgb as control.

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