Team:Uppsala/Project Sensing

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document.getElementById("tab2").innerHTML = '<h2>Method</h2><p>The first thing that needed to be done was to test if the system could work properly in E. coli. To test this we created three different versions of the yenbox: two fused with the Andersen promoters, J23113 and J23101 and one with the wildtype promoter, as this was most likely to interact properly with the yenbox. To be able to measure changes in the expression level we then attached the reporter gene BFP (Blur Fluorescence Protein) to each different design of the yenbox and then used a Fluorescence-activated cell sorting machine (FACS) to measure the median fluorescence from the cells in one overnight. This would then give us the base level of expression.<br></br>We then added the gene coding for YenR coupled to three promoters in different strength, to achieve different concentrations of YenR. The expression level will then be induced and the induction can be measured by comparing the expression with the previous one. Lastly, we added Yersinias signaling molecule to the sample, to see if we could inactive all the YenR and make the expression level return to its base rate.</p>';
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document.getElementById("tab2").innerHTML = '<p>While stealing the yenbox together with the wild type promoter fused with it, we had no idea about the strength of the wild type promoter or if it would even work at all. Because of this, we created an alternative version where we replaced the wild type promoter with a standardised one (J23113). In the wild type version there is an overlap between the wild type promoter and the yenbox. Hence, we mimicked the same while creating our customized version where we had an overlap between the promoter and the yenbox. Since the strength of the promoter would correspond to the leakage in our system, we wanted to have a weak promoter to minimize the leakage. We chose the constitutive promoter J23113 (BBa_J23113) from the Anderson library. Unfortunately, the promoter J23113 did not begin with the same two bases as the end of the yenbox. We were left with the option of either changing the two bases in the yenbox sequence or changing the two bases in the sequence of the promoter J23113. In the article by Ching-Sung Tsai and Stephen C. Winanas [1] they discovered that the binding between the activator YenR and the recognition region of the yenbox is not dependent on the entire sequence of the yenbox. Depending on which part of the yenbox is changed or replaced, YenR binds to the yenbox with different strengths. However, it still interacts with the yenbox and induces the strength of the promoter. Based on this fact, together with the knowledge that the Anderson promoters are very sequence dependent, we chose to change two bases in the sequence of the yenbox.<br><br>We also stole the coding sequence [2] of the activator YenR, from Y. enterocolitica, which we codon optimized for E. coli using a web tool [3] and synthesized it together with the RBS B0034 (BBa_B0034). Since we always want production of the activator YenR, it was coupled to three different constitutive promoters from the Anderson promoter library with three different strengths.</p>';
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Revision as of 16:30, 5 October 2014

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