Difference between revisions of "Team:SKLMT-China/Composite Part"

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             <table><thead><tr><td>Biobrick</td><td>Type</td><td>Description</td><td>length</td></tr></thead><tbody><tr><td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2569031">BBa_K2569031</a></td><td>Composite</td><td>PdnaA -NicA2</td><td>1525</td></tr><tr><td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2569032">BBa_K2569032</a></td><td>Composite</td><td>PdnaA-mRFP</td><td>782</td></tr></tbody></table><br/></p>
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             <table><thead><tr><td>Biobrick</td><td>Type</td><td>Description</td><td>length</td></tr></thead><tbody><tr><td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2569031">BBa_K2569031</a></td><td>Composite</td><td>PdnaA -NicA2</td><td>1525</td></tr><tr><td><a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2569032">BBa_K2569032</a></td><td>Composite</td><td>PampC-mRFP</td><td>782</td></tr></tbody></table><br/></p>
  
 
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Revision as of 21:22, 17 October 2018

Overview

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.

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.

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.

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

BiobrickTypeDescriptionlength
BBa_K2569031CompositePdnaA -NicA21525
BBa_K2569032CompositePampC-mRFP782