Our submitted parts include a promoter library of inner promoters with various strength built in
We used SDS-PAGE to demonstrate the expression of NicA2 (52.5 kDa). In the picture, we can see the clear expression of NicA2 in P.pf-5 containing plasmid with our promoter. It can be qualitatively known that our promoters can initiate transcription normally, and the nicotine-degrading gene cluster has also been successfully heterologously expressed.
As for transcription, we are going to use real-time PCR to compare the ability of initiating transcription of three promoters. In addition, we are comparing the degradation efficiency by HPLC. NicA2 in can convert nicotine to pseudo-oxidation in a whole-cell reaction, and the rest of the gene cluster will convert pseudo-oxidation to 2,5-DHP.
However, due to time limitation, we were unable to complete the last two experiments, but we will do a supplementary explanation in our presentation. Please stay tuned.