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| − | <header class="interlab-header"> | + | |
| | + | <header class="home-landing"> |
| | <div class="container"> | | <div class="container"> |
| − | <h1 class="display">InterLab</h1> | + | <h1 class="display">Stay tuned</h1> |
| | + | <p>We're working on something <b>amazing</b></p> |
| | </div> | | </div> |
| | </header> | | </header> |
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| − | <div class="content-wrapper under-nav text-layout"> | + | <div class="content-wrapper"> |
| − | <div class="container"> | + | <section class="container"> |
| − | | + | <h1>Project Description</h1> |
| − | <div class="row"> | + | |
| − | <div class="col-sm-8">
| + | In light of the recent discussions regarding the realisation of the UN development goal number 14 concerning the conservation and sustainable use of marine resources, iGEM Lund has set out to aid in the world-wide engagement against microplastic pollution. Microplastic pollution is an ever-growing problem that stems from wasteful lifestyles with propagating adverse effects throughout the entire food chain. Upwards of 10 million metric tons of plastic has been estimated to enter the ocean every year. Participating in the iGEM competition for the first time, team Lund intends to rapidly and accurately determine the presence of microplastics through the design and implementation of a genetic circuit into E. coli. A logic AND-gate will be constructed utilising the conformational change of a heterologously expressed hER-alpha to associate a split fluorescent reporter. |
| − | <section>
| + | </p> |
| − | <h3>Introduction</h3>
| + | </section> |
| − | <p> Since synthetic biology is built on engineering principles, reproducibility of the experiments and reliability of its standard parts are crucial for building predictable biological machines. This year’s InterLab study required all participants to produce comparable units for measuring the fluorescence of green fluorescence protein (GFP) following the same protocol to test the reproducibility of the experiment. Eight devices encoding GFP under three different promoters as well as two types of ribosome binding sites (RBS) were tested for their reliable gene expression in <em>Escherichia coli</em>.</p> | + | </div> |
| − | </section>
| + | |
| − | <section>
| + | |
| − | <h3>Materials and Methods</h3>
| + | |
| − | <ul>
| + | |
| − | <li>
| + | |
| − | Eight devices inserted in pSB1C3 provided by iGEM Headquarters:
| + | |
| − | <ul>
| + | |
| − | <li>Positive Control (<a href="http://parts.igem.org/Part:BBa_I20270" target="_blank">BBa_I20270</a>)</li>
| + | |
| − | <li>Negative Control (<a href="http://parts.igem.org/Part:BBa_R0040" target="_blank">BBa_R0040</a>)</li>
| + | |
| − | <li>Test Device 1 (<a href="http://parts.igem.org/Part:BBa_J364000" target="_blank">BBa_J364000</a>)</li>
| + | |
| − | <li>Test Device 2 (<a href="http://parts.igem.org/Part:BBa_J364001" target="_blank">BBa_J364001</a>)</li>
| + | |
| − | <li>Test Device 3 (<a href="http://parts.igem.org/Part:BBa_J364002" target="_blank">BBa_J364002</a>)</li>
| + | |
| − | <li>Test Device 4 (<a href="http://parts.igem.org/Part:BBa_J364003" target="_blank">BBa_J364003</a>)</li>
| + | |
| − | <li>Test Device 5 (<a href="http://parts.igem.org/Part:BBa_J364004" target="_blank">BBa_J364004</a>)</li>
| + | |
| − | <li>Test Device 6 (<a href="http://parts.igem.org/Part:BBa_J364005" target="_blank">BBa_J364005</a>)</li>
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − | <li>Expression host:<em> E. coli</em> DH5-α</li>
| + | |
| − | <li>Microtiter plate: 96 well plate, transparent with round base, Sarstedt </li>
| + | |
| − | <li>Plate reader for fluorescence measurements: Fluoroskan Ascent, Thermo Labsystems </li>
| + | |
| − | <li>Plate reader for OD595 measurements: Multiskan ascent, Thermo Electron Corporation</li>
| + | |
| − | </ul>
| + | |
| − | | + | |
| − | <table class="table table-bordered">
| + | |
| − | <thead>
| + | |
| − | <tr>
| + | |
| − | <th>Test device</th>
| + | |
| − | <th>Promoter</th>
| + | |
| − | <th>Promoter strength</th>
| + | |
| − | <th>RBS</th>
| + | |
| − | </tr>
| + | |
| − | </thead>
| + | |
| − | <tbody>
| + | |
| − | <tr>
| + | |
| − | <td>Negative control</td>
| + | |
| − | <td>TetR repressible promoter</td>
| + | |
| − | <td>medium</td>
| + | |
| − | <td>-</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Positive control </td>
| + | |
| − | <td>J23151</td>
| + | |
| − | <td> </td>
| + | |
| − | <td>B0032</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>1</td>
| + | |
| − | <td>J23101</td>
| + | |
| − | <td>1791</td>
| + | |
| − | <td>B0034</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>2</td>
| + | |
| − | <td>J23106</td>
| + | |
| − | <td>1185</td>
| + | |
| − | <td>B0034</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>3</td>
| + | |
| − | <td>J23117</td>
| + | |
| − | <td>162</td>
| + | |
| − | <td>B0034</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>4</td>
| + | |
| − | <td>J23101</td>
| + | |
| − | <td>1791</td>
| + | |
| − | <td>J364100</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>5</td>
| + | |
| − | <td>J23106</td>
| + | |
| − | <td>1185</td>
| + | |
| − | <td>J364100</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>6</td>
| + | |
| − | <td>J23117</td>
| + | |
| − | <td>162</td>
| + | |
| − | <td>J364100</td>
| + | |
| − | </tr>
| + | |
| − | </tbody>
| + | |
| − | </table>
| + | |
| − | <p>All constructs were received in the pSB1C3 plasmid carrying the gene for chloramphenicol resistancy.</p>
| + | |
| − | <p>The provided InterLab protocol was followed to perform the measurements, as well as the protocol for transformation. Colony PCR, which was not a required part of the InterLab study, was performed using published protocol.</p>
| + | |
| − | </section>
| + | |
| − | | + | |
| − | <section>
| + | |
| − | <h3>Results</h3>
| + | |
| − | | + | |
| − | <p>All raw data from measurements can be found <a href="https://static.igem.org/mediawiki/2017/8/85/T--Lund--InterLab_20170928_Measurements.xlsx" target="_blank">here</a>. In order to quantify the fluorescence of the expressed GFP in test devices, a standard curve as in <em>fig 1</em>, using fluorescein was made. For the absorbance measurements a conversion factor was needed to convert absorbance data to standard OD595 measurements. For that purpose LUDOX-S40 was used as a single reference point.</p>
| + | |
| − | | + | |
| − | <p>The result from colony PCR showed the size of the insert was about 1200 bp for the six test devices as well as the positive control device (P), and about 400 bp for the negative control device (N). The negative control contained water instead of the colony PCR product. </p>
| + | |
| − | </section>
| + | |
| − | | + | |
| − | <section>
| + | |
| − | <h3>Discussion</h3>
| + | |
| − | <p>After several unsuccessful attempts of obtaining fluorescence readings, colony PCR was performed to ensure the incubated colonies contained the plasmid with the correct gene insert, <em>fig 2</em>. The gel results of the colony PCR products indicated the correct gene inserts were present, meaning the transformation was successful. Based on the promoter strengths, the following outcomes were expected:</p>
| + | |
| − | <p>Device 1 and 4 - high fluorescence signal and low OD595</p>
| + | |
| − | <p>Device 2 and 5 - medium high fluorescence signal and OD595</p>
| + | |
| − | <p>Device 3 and 6 - low fluorescence signal and high OD595</p>
| + | |
| − | <p>The relative results matched the expected outcome to a certain extent. However, the absolute values for absorbance and fluorescence were on a level of background noise. Low OD595 values could be explained by low competency of <em>E. coli </em>DH5-α cells. Low fluorescence values might be the due to questionable reliability of RBS and improper physical conditions of the cell. It should be noted that the cell growth on chloramphenicol plates was consistently low throughout the project. For an elaboration on this matter, see results.</p>
| + | |
| − | </section>
| + | |
| − |
| + | |
| − | </div>
| + | |
| − | <div class="col-sm-4">
| + | |
| − | <figure>
| + | |
| − | <a href="#" data-featherlight="https://static.igem.org/mediawiki/2017/b/b3/T--Lund--InterLab--Fl_std_curve.svg">
| + | |
| − | <img class="img-responsive center-block img-thumbnail" src="https://static.igem.org/mediawiki/2017/b/b3/T--Lund--InterLab--Fl_std_curve.svg">
| + | |
| − | </a>
| + | |
| − | <figcaption class="text-center">Figure 1: Standard curve with fluorescein (μM).</figcaption>
| + | |
| − | </figure>
| + | |
| − | <figure>
| + | |
| − | <a href="#" data-featherlight="https://static.igem.org/mediawiki/2017/d/d7/T--Lund--InterLab--AGE_colony_PCR_170923.jpg">
| + | |
| − | <img class="img-responsive center-block img-thumbnail" src="https://static.igem.org/mediawiki/2017/d/d7/T--Lund--InterLab--AGE_colony_PCR_170923.jpg">
| + | |
| − | </a>
| + | |
| − | <figcaption class="text-center">Figure 2: Gel image from agarose gel electrophoresis with colony PCR samples.</figcaption>
| + | |
| − | </figure>
| + | |
| − | <figure>
| + | |
| − | <a href="#" data-featherlight="https://static.igem.org/mediawiki/2017/3/31/T--Lund--InterLab--Absorbance_plot-svg.svg">
| + | |
| − | <img class="img-responsive center-block img-thumbnail" src="https://static.igem.org/mediawiki/2017/3/31/T--Lund--InterLab--Absorbance_plot-svg.svg">
| + | |
| − | </a>
| + | |
| − | <figcaption class="text-center">Figure 3: Absorbance measurements of the devices at 0h, 2h, 4h and 6h.</figcaption>
| + | |
| − | </figure>
| + | |
| − | <figure>
| + | |
| − | <a href="#" data-featherlight="https://static.igem.org/mediawiki/2017/a/a3/T--Lund--InterLab--Fluorescence_plot.svg">
| + | |
| − | <img class="img-responsive center-block img-thumbnail" src="https://static.igem.org/mediawiki/2017/a/a3/T--Lund--InterLab--Fluorescence_plot.svg">
| + | |
| − | </a>
| + | |
| − | <figcaption class="text-center">Figure 4: Fluorescence measurements of the devices at 0h, 2h, 4h and 6h.</figcaption>
| + | |
| − | </figure>
| + | |
| − | </div>
| + | |
| − | </div> | + | |
| − | | + | |
| − | </div> | + | |
| − | </div> | + | |
| | | | |
| | </html> | | </html> |
| | {{Lund/footer}} | | {{Lund/footer}} |
Project Description
In light of the recent discussions regarding the realisation of the UN development goal number 14 concerning the conservation and sustainable use of marine resources, iGEM Lund has set out to aid in the world-wide engagement against microplastic pollution. Microplastic pollution is an ever-growing problem that stems from wasteful lifestyles with propagating adverse effects throughout the entire food chain. Upwards of 10 million metric tons of plastic has been estimated to enter the ocean every year. Participating in the iGEM competition for the first time, team Lund intends to rapidly and accurately determine the presence of microplastics through the design and implementation of a genetic circuit into E. coli. A logic AND-gate will be constructed utilising the conformational change of a heterologously expressed hER-alpha to associate a split fluorescent reporter.
