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Team:BostonU/ProjectTandemPromoters
From 2014.igem.org
| Tandem promoters condense higher order logic gates into fewer transcriptional units. For this reason, our team decided to add tandem promoters to our current MoClo library. In cycle one, we aimed to follow a formalized method for constructing tandem promoters based on what is in the literature.
To keep the tandem promoters compatible with a four-part MoClo transcriptional unit, we created a new fusion site, K, with the sequence ‘ATGC’. For each promoter pair, the primers are designed such that one promoter is flanked with ‘A’ and ‘K’ fusion sites and the other has ‘K’ and ‘B’ fusion sites. The two promoters were then combined in a MoClo reaction to form a level 0 tandem promoter part in ‘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. The tandem promoters were then made into level one parts with a fluorescent marker and tested in pro-strain competent cells using the flow cytometer. After testing and confirming the method for creating tandem promoters, we will be able to increase our variety of tandem promoters. This will give us greater flexibility when we build more complex logic circuits. |
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