Difference between revisions of "Team:SKLMT-China/Composite Part"
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| + | <section class="article-banner" style="background-image: url(https://static.igem.org/mediawiki/2018/6/6e/T--SKLMT-China--parts-banner-2.jpg)"> | ||
| + | <div class="banner-content"> | ||
| + | <h2 class="title">Composite Part</h2> | ||
| + | <p class="content">Standard nicotine degradation biobrick</p> | ||
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| + | <li><a href="https://2018.igem.org/Team:SKLMT-China"><i class="fa fa-home">  Home</i></a></li> | ||
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| + |   Composite Part | ||
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| + | <li><a href="#s1" class="scrolly">Overview</a></li> | ||
| + | <li><a href="#s2" class="scrolly">PdnaA-NicA2</a></li> | ||
| + | <li><a href="#s3" class="scrolly">Composite Parts</a></li> | ||
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| − | <h2 class="title">Overview </h2> | + | <h2 class="title" id="s1">Overview </h2> |
| − | <p>Our submitted parts include a promoter library of inner promoters with various strength built in <latin>Pseudomonas fluorescence</latin>-pf5. We also include well-characterized composite part. One includes our strongest promoter and nicotine oxidase NicA2, working well in | + | <p>Our submitted parts include a promoter library of inner promoters with various strength built in <latin>Pseudomonas fluorescence</latin>-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. </p> |
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| − | <h2 class="title"> | + | <h2 class="title" id="s2">PdnaA-NicA2 </h2> |
| − | < | + | <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> |
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| + | <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> | ||
| − | + | <p>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.</p><p> 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.</p> | |
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| + | <h2 class="title" id="s3">Composite Parts</h2> | ||
| + | <p></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><i class="fa fa-star" style="color: blue"></i><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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Latest revision as of 21:40, 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 | PampC-mRFP | 782 |