Team:HokkaidoU Japan/Promoter/Methods

From 2013.igem.org

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<h1>Method</h1>
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<h2>Promoter family</h2>
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<div class="fig fig400">
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<img src="https://static.igem.org/mediawiki/2013/a/a8/HokkaidoU2013_promoter_Method-fig1.png">
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<div>Fig. 1</div>
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</div>
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<p>As our first step for constructing original promoter family, we synthesized theoretically ideal consensus sequence to bind &sigma; factor. This should ensure that promoter will form the most stable complex with &sigma; 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 fig400">
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<img src="https://static.igem.org/mediawiki/2013/8/87/HokkaidoU2013_promoter_Method-fig2.png">
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<div>Fig.2</div>
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</div>
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<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]. We designed reverse promoter, promoter isolation primer, that is to isolate randomized promoter by annealing downstream of it [Fig.4].</p>
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<div class="fig fig400 para">
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<img src="https://static.igem.org/mediawiki/2013/5/5f/HokkaidoU2013_promoter_Method-fig3.png">
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<div>Fig.3</div>
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</div>
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<div class="fig fig400 para">
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<img src="https://static.igem.org/mediawiki/2013/7/78/HokkaidoU2013_promoter_Method-fig4.png">
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<div>Fig.4</div>
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</div>
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<h2>Assay</h2>
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<p>
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To measure transcription activities, we prepared two popular reporter genes and one antibiotics resistance gene, mRFP1, lacZ&alpha ;, and Kanamycin resistance gene.
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</p>
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Revision as of 01:25, 28 September 2013

Maestro E.coli

Promoter

Method

Promoter family

Fig. 1

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.2

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].

Fig.3
Fig.4

Assay

To measure transcription activities, we prepared two popular reporter genes and one antibiotics resistance gene, mRFP1, lacZ&alpha ;, and Kanamycin resistance gene.

Promoter Top
Results

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