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| | <hr class="section-heading-spacer"> | | <hr class="section-heading-spacer"> |
| | <div class="clearfix"></div> | | <div class="clearfix"></div> |
| − | <h2 class="section-heading">Experiments</h2>
| |
| | <p class="lead"> | | <p class="lead"> |
| − | <strong>Amplification of genes:</strong>
| + | To make sure we perform our experiments consistently, and that they can be reproduced by other scientists, we have written protocols for the experiments we have performed across the three subprojects. We have collected them all together below: |
| − | <ul style="text-align:left; color:white;">
| + | |
| − | <li>Synthetically produce a codon-optimised version of yddG. </li>
| + | |
| − | <li>Amplify trpE and aroG from WT E.coli MG1655</li>
| + | |
| − | <li> Point mutations for feedback resistance in trpE and aroG: Using primers with overhangs containing point mutations, and splitting the gene in two, then combining the two parts when inserting in the expression vector. </li>
| + | |
| − | <li> Created vector with combinations of one, two and three genes in the USER casette, using primers with overhangs.</li>
| + | |
| − | </ul>
| + | |
| − | </p>
| + | |
| − | <p class="lead">
| + | |
| − | To make the point mutations for trpE and aroG, two sets of vectors for each gene was designed (illustration). Overhangs in the end of the primers enable USER cassette insertion, while the primer overhangs in the center contain a point mutation. When the two parts are being amplified individually, the transformation into vector in expression host will be done with USER ligation. | + | |
| | <br><br> | | <br><br> |
| − | <strong>Vector design</strong> | + | <strong>General (verifications and vectors)</strong> |
| − | <br><br>
| + | |
| − | Protein import. USER casette and His tag. <br>
| + | |
| − | Vector design was performed in the protein import subproject, and the same vector was used for all cloning in the interdependency project.
| + | |
| − | <br><br>
| + | |
| − | <strong>Expression and production</strong>
| + | |
| | <ul style="text-align:left; color:white;"> | | <ul style="text-align:left; color:white;"> |
| − | <li>Expression checked with Western blotting: all genes HIS tagged. </li> | + | <li> Protocol 1: PCR </li> |
| − | <li>Tryptophane production by E.coli with one, two or three genes, and nuder different levels of inducing agents evaluated on HPLC</li> | + | <li>Protocol 2: Gel electrophoresis</li> |
| | + | <li> Protocol 3: Miniprep (homemade buffers) </li> |
| | + | <li> Protocol 4:Miniprep (E.Z.N.A)</li> |
| | </ul> | | </ul> |
| | </p> | | </p> |
| − | <p class="lead">
| |
| − | <strong>Co-growth of E.coli and yeast</strong>
| |
| − | <ul style="text-align:left; color:white;">
| |
| − | <li>Find yeast minimal media where E.coli can grow</li>
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| − | <li>Grow yeast in same media after E.coli has grown, in order to establish possibility for relationship.</li>
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| − | <li>Grow tryptophane producing E.coli for different time periods, then remove them and grow auxotrophic yeast in the media containing the produced tryptophane. </li>
| |
| − | </ul>
| |
| − | </p>
| |
| − | <p class="lead">
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| − | <strong>Growth of E.coli and yeast in same minimal yeast medium</strong>
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| − | <br><br>
| |
| − | E. coli strains MG1655 and BL21 were grown in several media in order to find a minimal yeast media where E.coli could survive. With inspiration from (van Summeren-Wesenhagen and Marienhagen, 2014), we decided to grow E.coli in the minimal yeast media YNB with the pH adjusted to 7 instead of 4 as original.
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| − | <br>
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| − | After ON growth of E.coli in YNB pH 7, the media was cleared of E.coli by spinning and filtration, after which it was inoculated with yeast (AM94), to ensure that the E.coli does not produce substances hindering yeast growth (protocol).<br>
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| − | This experiment is a prerequisite for our next experiment.
| |
| − | <br><br>
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| − | <strong>Growth of tryptophan auxotrophic yeast in minimal medium subsequent to tryptophan producing E.coli</strong>
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| − | <br><br>
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| − | This experiment utilizes the same protocol as the previous (protocol), but now in YNB pH7 media without a tryptophan source, with tryptophan overproducing E.coli and with a tryptophan auxotrophic yeast strain.This experiment is performed with single, double and triple transformations: That is, E.coli with trpE(fbr), aroG(fbr) and yddG alone or in combinations. The growth of yeast is measured using OD600 measurements to evaluate the successful complementation of the yeast amino acid auxotrophy by E.coli tryptophan production.
| |
| − | </p>
| |
| | </div> | | </div> |
| | </div> | | </div> |
