In this project, to carry out the aerobic transformation of Clostridium tyrobutyricum, the thlA promoter was modified to insert FNR binding site before the -35 region, making the promoter become an oxygen inhibition promoter. At first, we referred to the references and determined the distance between the FNR binding site and the -35 region as seven nucleotides, but the effect was not satisfying. Therefore, we contacted the author of this paper via email, Professor Zhou Shenghu of Jiangnan University. He put forward many constructive suggestions for our experiments. From his reply, we realized that our original design might not be a good choice. Then we adopted his suggestion about optimizing the promoter by adjusting the distance. In his paper, all modified promoters, which shortened the distance between the FNR binding site and the -35 region, all have a trend toward aerobic inhibition. Hence we proposed that the FNR on our promoter has the same trend. Then, we shortened the distance between the FNR binding site and the -35 region and remodeled the promoter to make thlA promoter gain aerobic inhibition. The new promoter showed the effect of aerobic inhibition in subsequent tests, which works as desired. What’s more, we are interested in the method of adding multi-copy of FNR binding sites up-stream of the -35 box, which was mentioned in the reply. We shared this design and our experience with Worldshaper_Nanjing.

Detailed experimental contents are shown in the link.