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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>';
-document.getElementById("tab3").innerHTML = '<p>For characterisation we created the constructs BBa_K1381008 and BBa_K1381009, were the yenbox fused with a promoter was coupled to the green fluorescent protein (GFP). These constructs were then cloned into the backbones pSB1C3 and pSB3C17 and transformed into competent E. coli cells already containing one of the YenR constructs BBa_K1381005, BBa_K1381006 or BBa_K1381007 on the backbone pSB1K3. The double transformed cells were then streaked on plates containing both the antibiotic Kanamycin and Chloramphenicol and left it overnight to grow. Cells containing only the constructs yenbox_promoter-B0032-GFP were also streaked and left to grow.<br><br>The following day, overnight cultures were prepared and left for 16 h to grow into stationary phase. After that, 10 µL of the overnight culture was put into 500 µL of PBS solution and left for one hour for stabilization. The green fluorescence production was then measured using a flow cytometer. The results of the test is shown below.<br><br>In graph 1 we can observe the induction but cannot predict to what extent. This is because these cells are transformed with two high copy plasmids containing the same ori(origin of replication). The consequence will be that the cells will confuse the two plasmids with each other and have no control of in what amount the two respective plasmids are present. It will only ensure that it is 100-300 plasmids present in total, regardless of which plasmid it is. This is why we also cloned the yenbox containing constructs into a low copy plasmid with a different ori than the high copy ones.<br>
+document.getElementById("tab3").innerHTML = '<p>For characterisation we created the constructs BBa_K1381008 and BBa_K1381009, were the yenbox fused with a promoter was coupled to the green fluorescent protein (GFP). These constructs were then cloned into the backbones pSB1C3 and pSB3C17 and transformed into competent E. coli cells already containing one of the YenR constructs BBa_K1381005, BBa_K1381006 or BBa_K1381007 on the backbone pSB1K3. The double transformed cells were then streaked on plates containing both the antibiotic Kanamycin and Chloramphenicol and left it overnight to grow. Cells containing only the constructs yenbox_promoter-B0032-GFP were also streaked and left to grow.<br><br>The following day, overnight cultures were prepared and left for 16 h to grow into stationary phase. After that, 10 µL of the overnight culture was put into 500 µL of PBS solution and left for one hour for stabilization. The green fluorescence production was then measured using a flow cytometer. The results of the test is shown below.<br><br>In graph 1 we can observe the induction but cannot predict to what extent. This is because these cells are transformed with two high copy plasmids containing the same ori(origin of replication). The consequence will be that the cells will confuse the two plasmids with each other and have no control of in what amount the two respective plasmids are present. It will only ensure that it is 100-300 plasmids present in total, regardless of which plasmid it is. This is why we also cloned the yenbox containing constructs into a low copy plasmid with a different ori than the high copy ones.<br>In graph 2, it can be seen that the amount of YenR that is produced is correlated, as expected, to the rate of the induction. When coupled to the strongest of the three promoters, J23102, the production is increased up to five folds.<br><br></p>';
-In graph 2, it can be seen that the amount of YenR that is produced is correlated, as expected, to the rate of the induction. When coupled to the strongest of the three promoters, J23102, the production is increased up to five folds.<br><br></p>';
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Team:Uppsala/Project Sensing

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Revision as of 16:33, 5 October 2014

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