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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> | ||
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| + | <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> | ||
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<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>Fig. 1</div> | <div>Fig. 1</div> | ||
</div> | </div> | ||
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| + | <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 [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>Fig.2</div> | <div>Fig.2</div> | ||
</div> | </div> | ||
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<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"> | ||
Revision as of 02:13, 28 September 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.
