Team:SKLMT-China/Composite Part


Our submitted parts include a promoter library of inner promoters with various strength built in Pseudomonas fluorescence-pf5. We also include well-characterized composite part. One includes our strongest promoter and nicotine oxidase NicA2, working well in enhancing nicotine degradation compared to original nicA2 gene.


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 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.

Whole protein SDS-PAGE electrophoresis: Expression of NicA2 (52.5kDa, showed in red) in pBBR1-km-amp-cm-nic (ck), pBBR1-km-amp-cm-promoter4-nic (promoter4), pBBR1-km-amp-cm-promoter5-nic (promoter5) and pBBR1-km-amp-cm-promoter11-nic (promoter11) in

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.

Composite Parts

BBa_K2569031CompositePdnaA -NicA21525