Difference between revisions of "Team:SKLMT-China/Composite Part"
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<p>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 <i>P.</i>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.</p> | <p>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 <i>P.</i>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.</p> | ||
<span class="image fit"> | <span class="image fit"> | ||
| − | <img src="https://static.igem.org/mediawiki/2018/e/ea/T--SKLMT-China--demonstrationfig2.png" alt=" | + | <img src="https://static.igem.org/mediawiki/2018/e/ea/T--SKLMT-China--demonstrationfig2.png" alt="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 P.pf-5. " /> |
</span> | </span> | ||
Revision as of 11:41, 17 October 2018
Overview
Our submitted parts include a promoter library of inner promoters with various strength built in
PdnaA-NicA2
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.
Composite Parts
| Biobrick | Type | Description | length |
| BBa_K2569031 | Composite | PdnaA -NicA2 | 1525 |
| BBa_K2569032 | Composite | PdnaA-mRFP | 782 |