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| − | <h4>Vector design</h4>
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| − | <p class="lead">
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| − | A modified version of the <a href="https://www.addgene.org/vector-database/2623/">pET102 vector</a> was used for expression of all genes in this sub-project. See <a href="https://2017.igem.org/Team:UCopenhagen/Protein-Import">protein import</a> for details on design and creation of the vector.<br></p>
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| − | <h4>Codon optimization and synthetic <i>yddG</i></h4>
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| − | <p class="lead">
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| − | Following problems with amplifying <i>yddG</i> from the genomic DNA, we decided to order a synthetically made version, and codon optimized it in the process.<br></p>
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| − |
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| − | <h4>Point mutations for feedback resistance in <i>trpE</i> and <i>aroG</i></h4>
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| − | <p class="lead">
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| − | We amplified the <i>aroG</i> and <i>trpE</i> genes using genomic DNA from <i>E.coli</i> strain MG1655. The amplification was done using two sets of primers per gene, with overhangs allowing for insertion of a point mutation in each gene and for cloning with USER assembly. <br><br>
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| − |
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| − | The mutations were designed in accordance with previous work by <u><a href="#I2">Gu <i>et al.</i> (2012)</a></u>. In TrpE, position 293 is changed from methionine to threonine, and for AroG position 150 is changed from proline to leucine.
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| − | <figure>
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| − | <br><br>
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| − | <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/5/5d/TrpE_pointmutation.png" alt="" width="250" height="200">
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| − | <br>
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| − | <figcaption><b>Figure 2 </b>Point mutation in <i>trpE</i>, and insertion in vector with USER assembly. To perform the point mutation, we designed two sets of primers for each gene. The outer primers are the same as used for amplification from the genomic DNA. The central primers have overhangs containing the point mutations.
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| − | This way, the genes are split in two, and the two parts are combined when inserting in the USER cassette in the expression vector - and now containing a point mutation in the middle.
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| − | </figcaption>
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| − | </figure>
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| − | </p>
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| − | <h4>Multi-gene constructs</h4>
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| − | <p class="lead">
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| − | To evaluate the effect of the proteins, we made multi-gene constructs.
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| − | The constructs were made by designing primers with overhangs encoding Ribosome Binding Sites (RBS). RBS makes sure all genes will be transcribed, and the overhangs enable combination of the genes into multi-gene constructs (fig 3) and USER assembly.<br><br>
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| − | We created biobricks of one or two genes in combination. We attempted to make all the following constructs into biobricks, but did not succeed with the 3 gene construct.
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| − | <ul style="text-align:left; color:white;">
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| − | <li>YddG-his</li>
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| − | <li>TrpE-his</li>
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| − | <li>AroG-his</li>
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| − | <li>YddG-TrpE-his: <a href="http://parts.igem.org/Part:BBa_K2455007">BBa_K2455007</a></li>
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| − | <li>YddG-AroG-his: <a href="http://parts.igem.org/Part:BBa_K2455008">BBa_K2455008</a></li>
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| − | <li>YddG-TrpE-AroG-his</li>
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| − | </ul>
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| − | </p>
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| − |
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| − | <p class="lead">
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| − | <figure>
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| − | <br><br>
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| − | <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/f/ff/Construct_insertions2.png" alt="" width="250" height="200">
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| − | <br>
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| − | <figcaption><b>Figure 3 </b>Multi-gene constructs were created with a series of primers with overhangs coding for Ribosome Binding Sites (RBS) between the genes. The outer primers coded for USER overhangs for USER ligation.
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| − | </figcaption>
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| − | </figure>
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| − | </p>
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| − |
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| − | <h4>Serial growth experiment of <i>E.coli</i> and yeast</h4>
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| − | <p class="lead">
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| − | <i>E. coli</i> strains MG1655 and BL21 were grown in several media, until we settled on the minimal yeast media YNB with the pH adjusted to 7 (<u><a href="#I1">van Summeren-Wesenhagen and Marienhagen, 2014</a></u>). <br><br>
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| − | To ensure that <i>E.coli</i> do not produce substances hindering yeast growth, we performed an initial serial growth experiment, shown in figure 4. The serial growth experiment was performed with ON growth of <i>E.coli</i> in YNB pH 7, clearing the media of E.coli by spinning and filtration through 0.2 µm filters, and inoculating with yeast (AM94).
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| − | <figure>
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| − | <br><br>
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| − | <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/4/42/Serialgrowth1.png" alt="" width="250" height="200">
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| − | <br>
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| − | <figcaption><b>Figure 4 </b>Serial growth experiment. 1) E.coli from liquid culture in LB media is inoculated in YNB media. 2) After sufficient growth of E.coli in YNB, the media is cleared of E.coli by spinning and filtration. 3) E.coli free media is then inoculated with tryptophan auxotroph yeast.
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| − | </figcaption>
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| − | </figure>
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| − | </p>
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| − |
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| − | <h4>Serial growth experiment with transformed <i>E.coli</i></h4>
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| − | <p class="lead">
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| − | To evaluate, if the complementation of the yeast amino acid auxotrophy by <i>E.coli</i> tryptophan production is successful, we made serial growth experiments with transformed <i>E. coli</i>. <br><br>
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| − | We first growed the E.coli, transformed with the single, double and triple construct vectors described above in <a href="https://static.igem.org/mediawiki/2017/f/f6/Protocol18_UCopenhagen.pdf">YNB media</a> without a tryptophane source. Subsequently, we cleared the media and inoculated it with auxotroph yeast, then evaluated the growth using OD<sub>600</sub> measurements. <br><br>
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| − |
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| − | We also checked the growth of auxotrophic yeast in the media with and without added tryptophan, to ensure that any growth after <i>E.coli</i> growth was due to tryptophan being produced by <i>E.coli</i> and not due to the yeast overcoming the auxotrophy.
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| − | All serial growth experiments used variations of the same <a href="https://static.igem.org/mediawiki/2017/0/0e/Protocol19_UCopenhagen.pdf">protocol</a>.
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| − | </p>
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| − |
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| − | <div class="col-lg-5 col-sm-6">
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| − | <hr class="section-heading-spacer">
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| − | <div class="clearfix"></div>
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| − |
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| − | <h4>HPLC measurements of tryptophan production</h4>
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| − | <p class="lead">
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| − | In addition to the qualitative assessment of tryptophan production by yeast growth, we made a quantitative evaluation of tryptophan production by our transformed <i>E.coli</i>. <br><br>
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| − | We used <a href="https://static.igem.org/mediawiki/2017/b/b8/Protocol20_UCopenhagen.pdf">HPLC-MS </a> with a program for amino acid detection to measure the concentration of tryptophan in the media after 10-40 hours growth. Internal and external standards were used. Simultaneously, we measured concentrations of tyrosine and phenylalanine, which should be increased when AroG is expressed. <br><br>
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| − |
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| − | We measure production from the following transformations of E. coli.
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| − | <ul style="text-align:left; color:white;">
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| − | <li>empty vector</li>
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| − | <li>YddG-his</li>
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| − | <li>TrpE-his </li>
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| − | <li>AroG-his</li>
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| − | <li>YddG-TrpE-his</li>
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| − | <li>YddG-AroG-his</li>
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| − | <li>YddG-TrpE-AroG-his</li>
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| − | <li>YddG-TrpE-AroG</li>
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| − | </ul>
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| − | </p>
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| − | <p class="lead">Growth of these transformants are simultaneously monitored with OD<sub>600</sub> measurements. </p>
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| − | </div>
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| − |
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| − | <div class="col-lg-5 col-lg-offset-2 col-sm-6">
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| − | <br><br>
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| − | <br><br>
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| − | <figure>
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| − | <img class="img-responsive3" src="https://static.igem.org/mediawiki/2017/9/97/HPLC-MS-foto.jpg" alt="" width="250" height="200">
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| − | <br>
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| − | <figcaption><b>Figure 5 </b>Photo of the HPLC-MS set-up.
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| − | </figcaption>
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| − | </figure>
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| − | </div>
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| | </div> | | </div> |
| | </div> | | </div> |
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| | <div class="clearfix"></div> | | <div class="clearfix"></div> |
| | <h2 class="section-heading">Design process/future</h2> | | <h2 class="section-heading">Design process/future</h2> |
| − | <p class="lead">
| + | |
| − | <strong>Summary</strong>: We cloned <i>E.coli</i> strains to express proteins in the tryptophan synthesis pathway, and have measured the production of these cells. We have ensured that auxotroph yeast can grow after <i>E.coli</i> in the same media, and have attempted to grow it on the tryptophan produced by <i>E. coli</i> as the single tryptophan source.
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| − | <br><br>
| + | |
| − | In our design process, we have considered a wide range of possible gene combinations and interdependency mechanisms. We settled on a simple amino acid interdependency which is easy to test with an amino acid auxotroph yeast.
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| − | <br><br>
| + | |
| − | In a final version of an endosymbiotic relationship, amino acid dependency might not be the optimal dependency as it would not allow the system to be grown on a media with all amino acids that might be required for co-cultures for added modularity. Instead of tryptophan, a different, but vital metabolite could be supplied by the symbiont. <br><br>
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| − | To perform our tryptophan overproduction, we chose genes that, when over-expressed would have the greatest impact, based on the article by <u><a href="#I2">Gu <i>et al.</i> (2012)</a></u>. Initially, we considered simply overexpressing the tryptophan operon, but quickly realised this would be highly downregulated due to negative feedback regulation.<br><br>
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| − | To release the feedback regulation, we decided on an amino acid translocator to reduce the intracellular tryptophan concentration. Several deletions of e.g. endogenous trpR has also been considered, but this would make our project overly complicated due to the difficulty of making such deletions in <i>E.coli</i>.
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| − | </p>
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| | </div> | | </div> |
| | </div> | | </div> |
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| | <h2 class="section-heading">References</h2> | | <h2 class="section-heading">References</h2> |
| | <p class="lead"> | | <p class="lead"> |
| − | <a name="I1">Summeren-Wesenhagen, P. V., & Marienhagen, J. (2014). Metabolic Engineering of Escherichia coli for the Synthesis of the Plant Polyphenol Pinosylvin. <i>Applied and Environmental Microbiology, 81</i>(3), 840-849. doi:10.1128/aem.02966-14</a>
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| − | <br><br>
| + | |
| − | <a name="I2">Gu, P., Yang, F., Kang, J., Wang, Q., & Qi, Q. (2012). One-step of tryptophan attenuator inactivation and promoter swapping to improve the production of L-tryptophan in Escherichia coli. <i>Microbial Cell Factories, 11</i>(1), 30. doi:10.1186/1475-2859-11-30</a>
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| − | <br><br>
| + | |
| − | <a name="I3">Wang, J., Cheng, L., Wang, J., Liu, Q., Shen, T., & Chen, N. (2013). Genetic engineering of Escherichia coli to enhance production of l-tryptophan. <i>Applied Microbiology and Biotechnology, 97</i>(17), 7587-7596. doi:10.1007/s00253-013-5026-3</a>
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| − | <br><br>
| + | |
| − | <a name="I4">Sigma-Aldrich. (2017). Yeast Synthetic Drop-out Medium Supplements Y1501. (n.d.). Retrieved October 31, 2017, from http://www.sigmaaldrich.com/catalog/product/sigma/y1501?lang=en®ion=DK</a>
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| − | <br><br>
| + | |
| − | <a name="I5">Bianciotto, V., Lumini, E., Lanfranco, L., Minerdi, D., Bonfante, P., & Perotto, S. (2000). Detection and Identification of Bacterial Endosymbionts in Arbuscular Mycorrhizal Fungi Belonging to the Family Gigasporaceae. <i>Applied and Environmental Microbiology, 66</i>(10), 4503-4509. doi:10.1128/aem.66.10.4503-4509.2000</a>
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| − | <br><br>
| + | |
| − | <strong>Reference for USER cloning</strong>:
| + | |
| − | <br>
| + | |
| − | <a name="I6">Nour-Eldin, H. H., Hansen, B. G., Nørholm, M. H., Jensen, J. K., & Halkier, B. A. (2006). Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. <i>Nucleic Acids Research, 34</i>(18). doi:10.1093/nar/gkl635</a>
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| − | <br><br>
| + | |
| − | <strong>Reference for USER fusion</strong>:
| + | |
| − | <br>
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| − | <a name="I7">Geu-Flores, F., Nour-Eldin, H. H., Nielsen, M. T., & Halkier, B. A. (2007). USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. <i>Nucleic Acids Research, 35</i>(7). doi:10.1093/nar/gkm106</a>
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| | </p> | | </p> |
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