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Background

Controlling the movement of our E.coli was essential for tracking down the pathogen. We chose to do so by controlling the expression of cheZ, a gene in the chemotactic pathway of E.coli. Without it, E.coli is immotile.

System design

A construct with an RBS, a promoter and cheZ was created and inserted into a cheZ-knockout strain to see if motility could be restored. Three promoters with different strengths were used to see how different expression levels of cheZ would affect motility. Finally, cheZ was to be coupled with the system created by the Sensing group.

Result

By inoculating our E.coli on swarm plates, we were able to observe if their motility had been restored. The motility of cells containing the construct was only restored in one of many experiments. Positive controls on the swarm plate assay indicated that results were often not reproducible, however.

Parts

Assembly Plan

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@@ Line 7: / Line 7: @@
 <div class="background">
 <a href="#ref_point" style="display:block; width:100%; height: 100%; text-decoration:none !important;"><h2 class="overview">Background</h2>
-<p class="box_text">In our project we developed a system to fight pathogens in the gut, which is not a unique idea. Except we wanted our systems key attribute to be specificity. To do so, we needed to find a unique quorum sensing language that we could hijack and utilize. We found the Yen system, from <i>Yersinia enterocolitica</i>. A homologous sytem to the well known Lux system, from <i>Vibrio fischeri</i>.</p>
+<p class="box_text">Controlling the movement of our <i>E.coli</i> was essential for tracking down the pathogen. We chose to do so by controlling the expression of <i>cheZ</i>, a gene in the chemotactic pathway of <i>E.coli</i>. Without it, <i>E.coli</i> is immotile.</p>
 </a>
 </div>
@@ Line 21: / Line 21: @@
 <h2 class="overview">System design</h2>
-<p class="box_text">From the Yen system we stole the activator YenR and the recognition region, the yenbox, fused with a wild type promoter. YenR was synthesized with the RBS B0034 and codon optimized for <i>E. coli</i>. A customized version of the yenbox-promoter was also created.</p>
+<p class="box_text">A construct with an RBS, a promoter and <i>cheZ</i> was created and inserted into a <i>cheZ</i>-knockout strain to see if motility could be restored. Three promoters with different strengths were used to see how different expression levels of <i>cheZ</i> would affect motility. Finally, <i>cheZ</i> was to be coupled with the system created by the Sensing group.</p>
 </a>
@@ Line 31: / Line 31: @@
 <h2 class="overview">Result</h2>
-<p class="box_text">We managed to get a five-fold induction relative to the base level of expression. The cells that were used during the measurements were double transformed with the yenbox-promoter complex with GFP and a plasmid producing the activator YenR.</p>
+<p class="box_text">By inoculating our <i>E.coli</i> on swarm plates, we were able to observe if their motility had been restored. The motility of cells containing the construct was only restored in one of many experiments. Positive controls on the swarm plate assay indicated that results were often not reproducible, however. </p>
 </a>
 </div>

Team:Uppsala/Project Targeting

From 2014.igem.org