Team:HokkaidoU Japan/Promoter/Methods
From 2013.igem.org
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Revision as of 01:26, 28 September 2013
Maestro E.coli
Promoter
![](https://static.igem.org/mediawiki/2013/e/ea/HokkaidoU2013_Maestro_Header.png)
Method
Promoter family
![](https://static.igem.org/mediawiki/2013/a/a8/HokkaidoU2013_promoter_Method-fig1.png)
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].
![](https://static.igem.org/mediawiki/2013/8/87/HokkaidoU2013_promoter_Method-fig2.png)
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].
![](https://static.igem.org/mediawiki/2013/5/5f/HokkaidoU2013_promoter_Method-fig3.png)
![](https://static.igem.org/mediawiki/2013/7/78/HokkaidoU2013_promoter_Method-fig4.png)
Assay
To measure transcription activities, we prepared two popular reporter genes and one antibiotics resistance gene, mRFP1, lacZ&alpha ;, and Kanamycin resistance gene.