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Team:HokkaidoU Japan/Promoter/Methods
From 2013.igem.org
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<h2>Promoter family</h2> | <h2>Promoter family</h2> | ||
| - | <p>As our first step for constructing original promoter family, we synthesized theoretically ideal consensus sequence to bind σ factor. This should ensure that promoter will form the most stable complex with σ factor. We synthesized such a consensus promoter showed in the figure above, originated from consensus sequence and lac operon promoter (pLac) | + | <p>As our first step for constructing original promoter family, we synthesized theoretically ideal consensus sequence to bind σ factor. This should ensure that promoter will form the most stable complex with σ factor. We synthesized such a consensus promoter showed in the figure above, originated from consensus sequence and lac operon promoter (pLac) (fig.1).</p> |
<div class="fig fig800"> | <div class="fig fig800"> | ||
<img src="https://static.igem.org/mediawiki/2013/a/a8/HokkaidoU2013_promoter_Method-fig1.png"> | <img src="https://static.igem.org/mediawiki/2013/a/a8/HokkaidoU2013_promoter_Method-fig1.png"> | ||
| - | <div> | + | <div>fig. 1</div> |
</div> | </div> | ||
| - | <p>We constructed consensus promoter by primer annealing | + | <p>We constructed consensus promoter by primer annealing(fig.2). |
| - | For mutating hexamer at -35 region, a promoter randomize primer which has random hexamer (NNNNNN) at -35 region was used, but other sequence in the primer is same with consensus promoter | + | For mutating hexamer at -35 region, a promoter randomize primer which has random hexamer (NNNNNN) at -35 region was used, but other sequence in the primer is same with consensus promoter(fig.3). We designed reverse promoter, promoter isolation primer, that is to isolate randomized promoter by annealing downstream of it(fig.4).</p> |
<div class="fig fig800"> | <div class="fig fig800"> | ||
<img src="https://static.igem.org/mediawiki/2013/8/87/HokkaidoU2013_promoter_Method-fig2.png"> | <img src="https://static.igem.org/mediawiki/2013/8/87/HokkaidoU2013_promoter_Method-fig2.png"> | ||
| - | <div> | + | <div>fig.2</div> |
</div> | </div> | ||
<div class="fig fig800 "> | <div class="fig fig800 "> | ||
<img src="https://static.igem.org/mediawiki/2013/5/5f/HokkaidoU2013_promoter_Method-fig3.png"> | <img src="https://static.igem.org/mediawiki/2013/5/5f/HokkaidoU2013_promoter_Method-fig3.png"> | ||
| - | <div> | + | <div>fig.3</div> |
</div> | </div> | ||
<div class="fig fig800 "> | <div class="fig fig800 "> | ||
<img src="https://static.igem.org/mediawiki/2013/7/78/HokkaidoU2013_promoter_Method-fig4.png"> | <img src="https://static.igem.org/mediawiki/2013/7/78/HokkaidoU2013_promoter_Method-fig4.png"> | ||
| - | <div> | + | <div>fig.4</div> |
</div> | </div> | ||
<div class="clearfix"></div> | <div class="clearfix"></div> | ||
Revision as of 07:47, 20 October 2013
Maestro E.coli
Promoter
Method
Promoter family
As our first step for constructing original promoter family, we synthesized theoretically ideal consensus sequence to bind σ factor. This should ensure that promoter will form the most stable complex with σ factor. We synthesized such a consensus promoter showed in the figure above, originated from consensus sequence and lac operon promoter (pLac) (fig.1).
We constructed consensus promoter by primer annealing(fig.2). For mutating hexamer at -35 region, a promoter randomize primer which has random hexamer (NNNNNN) at -35 region was used, but other sequence in the primer is same with consensus promoter(fig.3). We designed reverse promoter, promoter isolation primer, that is to isolate randomized promoter by annealing downstream of it(fig.4).
Assay
To measure transcription activities, we prepared two popular reporter genes and one antibiotics resistance gene, mRFP1, lacZα, and Kanamycin resistance gene.
