Team:SKLMT-China/Demonstrate

Demonstrate

Based on our promoter library, we selected three promoters (promoter 4, promoter 5 and promoter11) of different intensities to regulate the expression of the key nicotine-degrading gene nicA2.We’ve constructed lasmid, pBBR1-km-amp-cm-promoter-nic, and electroporated it into our new chassis bacteria, Pseudomonas fluorescences pf-5.

We found that there are only a few colonies of P. fluorescences pf-5 (containing nicA2 with promoters in front of it)on the LB plate,but lots of colonies on the CK plate(control group electroporated with pBBR1-km-amp-cm-nic without promoter). Combining the fact that theE.coliusually lost a part of (sometimes the whole plasmid)when we want to use high-copy vector to carry this gene cluster,we wondered if it NicA2 is a poisonous enzyme so that E.coli lost this plasmid during DNA replication under stress.

Plasmid in P. fluorescences pf-5

Strength of promoter

CFU on LB plate

pBBR1-km-amp-cm-nic

none

>200

pBBR1-km-amp-cm-promoter4-nic

normal

5

pBBR1-km-amp-cm-promoter5-nic

weak

5

pBBR1-km-amp-cm-promoter11-nic

strong

1

In order to test if our plasmids really work in P. fluorescences pf-5, we demonstrate our project in three levels, transcription, protein expression, and substrate degradation efficiency.

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. fluorescences pf-5 containing plasmid with promoter. It can be qualitatively known that our promoter 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 ofinitiating 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.

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