Team:BostonU/ProjectTandemPromoters

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Revision as of 21:59, 13 October 2014

Tandem Promoters

In order to build the priority encoder for phase III of Project Chimera, our team decided to add tandem promoters to our current MoClo library. Tandem promoters condense higher order logic gates into fewer transcriptional units and also allow two different inputs for one transcriptional unit. In phase 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:

pBad-pTet, pTet-pBad, pA1LacO-pTet, pA1LacO-pBad, pBad-pA1LacO, pTet-pA1LacO

These three promoters were easily accessible within our lab and are well understood. This summer, we completed the pBad-pTet and pTet-pBad promoters. After creating the tandem promoters, we transformed them into bioline competent cells and confirmed by sequencing. The tandem promoters were then made into level one parts with a fluorescent marker (RFP) and tested in pro-strain competent cells using the flow cytometer. The prostrain cells already have araC and tetR, which means the pTet-pBad and pBad-pTet promoters are turned off when there are no small molecules present. We were then able to add various concentrations of arabinose, atc, and both to obtain transfer curves.

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. New tandem promoters will be created using pLmrA, pSrpR, and pBetI.

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-         <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 phase one, we aimed to follow a formalized method for constructing tandem promoters based on what is in the literature.
+         <th scope="col">In order to build the priority encoder for phase III of Project Chimera, our team decided to add tandem promoters to our current MoClo library. Tandem promoters condense higher order logic gates into fewer transcriptional units and also allow two different inputs for one transcriptional unit. In phase one, we aimed to follow a formalized method for constructing tandem promoters based on what is in the literature.
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 <center><img src="https://static.igem.org/mediawiki/2014/b/b5/BostonU_Aug12_TandemPromoters.png"  alt="FP" ></center>
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 <center> pBad-pTet, pTet-pBad, pA1LacO-pTet, pA1LacO-pBad, pBad-pA1LacO, pTet-pA1LacO
 <br><br>
-  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.
+  These three promoters were easily accessible within our lab and are well understood. This summer, we completed the pBad-pTet and pTet-pBad promoters. After creating the tandem promoters, we transformed them into bioline competent cells and confirmed by sequencing. The tandem promoters were then made into level one parts with a fluorescent marker (RFP) and tested in pro-strain competent cells using the flow cytometer. The prostrain cells already have araC and tetR, which means the pTet-pBad and pBad-pTet promoters are turned off when there are no small molecules present. We were then able to add various concentrations of arabinose, atc, and both to obtain transfer curves.
 <br><br>
-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.
+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. New tandem promoters will be created using pLmrA, pSrpR, and pBetI.
 </th></tr>