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Team:BostonU/ProjectTandemPromoters
From 2014.igem.org
| Our team chose to make several tandem promoters to expand the range of circuits we can build and to develop a procedure for building tandem promoters. After reading several papers and discussing with the Voigt lab, we decided to combine two promoters without any extra sequences between them.
To make this compatible with MoClo, we created a new fusion site, K, with the sequence ‘ATGC’. For each promoter pair, one promoter is flanked with an ‘A’ and ‘K’ fusion site and the other has a ‘K’ and ‘B’ fusion site. The forward and reverse primers that we used to amplify the promoters each contained one of these fusion sites. The two promoters were then combined in a MoClo reaction to form a level 0 tandem promoter part in an ‘AB’ destination vector. In order to test this method, we used pBad, pTet, and pA1lacO promoters to form all possible combinations of hybrid promoters. These three promoters were easily accessible within our lab and are well understood. After creating the tandem promoters, they were transformed into bioline competent cells and confirmed by sequencing. After testing and confirming the method for creating tandem promoters, we will be able to increase the types of promoters. This will give us greater flexibility when we go to build more complex logic circuits. Tandem promoters will allow us to create larger logic circuits with fewer transcriptional units, which will hopefully decrease the amount of human error involved in building genetic circuits. |
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