Team:BostonU/ProjectTandemPromoters

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Revision as of 17:48, 12 August 2014

Tandem Promoters

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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-         <th scope="col">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.
+         <th scope="col">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.
 <br><br>
-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.
+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.
 <br><br>
-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.
+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.
 <br><br>
-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.
+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.
 </th></tr>