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| − | <a class="nav-link" href="#july">July 2017</a>
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| − | <a class="nav-link" href="#august">August 2017</a>
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| − | <a class="nav-link" href="#september">September 2017</a>
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| − | <h3 id="pageHeader">Experiments</h3>
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| − | <h4 id="july">July 2017</h4>
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| − | <h5>18<sup>th</sup> July</h5>
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| − | <p>A Taq polymerase PCR procedure was run to amplify the fim operon in E.coli strains MG1655 (as a reference) and DH5ɑ (if present). The primers were based on the known sequence for the operon and several were used in order to separate the three genes fimD, fimH and fimA into different bands on the subsequent electrophoresis. </p>
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| − | <h5>19<sup>th</sup> July</h5>
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| − | <p>The products of the PCR from the previous day were run down an agarose gel. As shown on the image, DH5ɑ contains all three of the fim genes which were tested for. If DH5ɑ were to be used as a chassis for our product, the fim genes would have had to have been knocked out prior to the insertion of the operon and modified fimH gene containing plasmids. The decision was made to look for an alternative strain of E.coli that does not contain the genes for pilus biosynthesis.</p>
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| − | <img class="rounded mx-auto d-block w-50" src="https://static.igem.org/mediawiki/2017/6/6e/T--Exeter--July_19th2.jpeg" alt="July 19th">
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| − | <h5>25<sup>th</sup> July</h5>
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| − | <p>With a genomic extraction having been done at the beginning of the day, another PCR was carried out with the same primers previously used in order to amplify any present fimA,D or H genes in MG1655(as a reference) and Top10. </p>
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| − | <h5>26<sup>th</sup> July</h5>
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| − | <p>An electrophoresis was carried out using the amplified DNA from the previous day. As shown, the MG1655 reference is positive for all threefim genes that were amplified, while Top10 is negative for all three. This confirmed that Top10 was a more suitable candidate with regards to its genome. </p>
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| − | <img class="rounded mx-auto d-block w-50" src="https://static.igem.org/mediawiki/2017/9/99/T--Exeter--July_26th2.jpeg" alt="July 26th">
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| − | <h5>28<sup>th</sup> July</h5>
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| − | <p>Overnight cultures of MG1655 and Top10 were removed from the incubator and were taken to the Bioimaging department. The samples were prepared with a negative stain and were placed on a small, circular copper grid before being inserted into the transmission electron microscope. Severalexposures were taken, as shown and these are demonstrated both that MG1655 does indeed produce pilus structures and that pili are absent from the surface of the Top10 strain. This, combined with the electrophoresis evidence, confirmed that the Top10 strain is the suitable chassis for our project. </p>
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| − | <h4 id="august">August 2017</h4>
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| − | <h5>2<sup>nd</sup> August</h5>
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| − | <p>The fim operon + pAra MoClo products were cut using restriction endonucleases before being run down an agarose gel. The result gave confirmation that the fim operon had been transformed wholly into dh5ɑ in pSB1A3.</p>
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| − | <img class="rounded mx-auto d-block w-50" src="https://static.igem.org/mediawiki/2017/c/cb/T--Exeter--August_2nd2.jpeg" alt="August 2nd">
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| − | <h5>31<sup>st</sup> August</h5>
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| − | <p>The first attempt at the yeast agglutination protocol was made. Overnight cultures of Saccharomyces cerevisiae were incubated at 30°C with varying volumes of Top10 WT and MG1655WT. They were initially kept in the shaking incubator and were then transferred to a water bath at the same temp overnight. The MG1655 samples demonstrated agglutination by showing a pellet, while the Top10 samples did not.</p>
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| − | <h4 id="september">September</h4>
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| − | <h5>1<sup>st</sup> September</h5>
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| − | <p>The first attempt at the yeast agglutination protocol was made. Overnight cultures of Saccharomyces cerevisiae were incubated at 30°C with varying volumes of Top10 WT and MG1655WT. They were initially kept in the shaking incubator and were then transferred to a water bath at the same temp overnight. The MG1655 samples demonstrated agglutination by showing a pellet, while the Top10 samples did not.</p>
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| − | <h5>6<sup>th</sup> September</h5>
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| − | <p>The solutions of induced bacterial cultures and metal ions (of varying concentrations) were prepared and pipetted into Snakeskin dialysis tubing. The tubing was clamped and suspended in a beaker of MOPS buffer solution. The beakers were placed in a shaking incubator for 1 hour at 20°C and 100rpm. After this period, the MOPS solution was removed and sampled for later ICPOES readings. The MOPS solution was replaced and the previous step was repeated twice. <br> The samples were run through the TECAN for fluorescence as done on the 1st.</p>
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| − | <h4 id="references">References</h4>
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