Team:HokkaidoU Japan/Promoter/Methods

From 2013.igem.org

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<p>We constructed consensus promoter by primer annealing (fig.2).
<p>We constructed consensus promoter by primer annealing (fig.2).
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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). Then, we selected 10 promoters according to assay result. We designed promoter isolation primer, that is to isolate randomized promoter by annealing downstream of it (fig.4).</p>
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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 transformed the promoter-randomized constructs. We could logically find 4096 kinds of colonies on the plate. Then, we selected 10 promoters according to assay result. We designed 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">

Revision as of 05:31, 27 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).

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 transformed the promoter-randomized constructs. We could logically find 4096 kinds of colonies on the plate. Then, we selected 10 promoters according to assay result. We designed promoter isolation primer, that is to isolate randomized promoter by annealing downstream of it (fig.4).

fig.2
fig.3
fig.4

Assay

To measure transcription activities, we prepared two popular reporter genes and one antibiotics resistance gene, mRFP1, LacZα, and Kanamycin resistance gene.