Difference between revisions of "Team:UCopenhagen/Parts"

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                         <h1>I N T E R  L A B</h1>
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                         <h1>P A R T S</h1>
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                     <h2 class="section-heading">Introduction </h2>
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                     <h2 class="section-heading">Cell measurements </h2>
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                     <p class="lead">Our team believes that establishing a stable platform for scientists to create naïve orthogonal living compartments, would allow for an unpredictable advancement in the field of synthetic biology. Our project will not attempt to create an endosymbiont, but instead investigate the mechanisms in free-living cells in a bottom-up approach to endosymbiosis.
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                     <p class="lead">Two colonies from each transformation were picked, and grown in foil-covered 50 ml falcon tubes over night (18 hours). </p>
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The endosymbiotic theory, formulated in the early years of the previous century, outlines that the organelles of the eukaryotic cell, such as the mitochondria, have their origin in free-living prokaryotes engulfed by bigger cells. These incorporated cells then co-evolved with their host conferring to it novel emergent properties which ultimately helped fuel the development of more complex multicellular biological systems such as plants and animals (Archibald, 2015). </p>
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<p class="lead"><strong>Preparation</strong> OD was measured, and a dilution was calculated to achieve an OD of 0.02. Dilution calculations can be found in the table next to this. Here we used the calculated correction factor from our initial abs/OD calibration. From the absorption measurements taken at 0 hours, we see indications of pipetting errors, as the OD<sub>600</sub> (average) ranges from 0.015 to 0.6 (table 1). </p>
  
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<button onclick="javascript:showhide('InterLab-table')">Show table 1</button>
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<div id="InterLab-table" style="display:none;">
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<div class="container">
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  <table class="responsive-table">
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    <caption><b>Table 1</b> Starting OD.</caption>
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    <thead>
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      <tr>
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<th scope="col"></th>
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        <th scope="col">Abs<sub>600</sub></th>
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        <th scope="col">OD<sub>600</sub> before</th>
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        <th scope="col">Volume (ml) added to 12 ml media</th>
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        <th scope="col">OD<sub>600</sub> after dilution (0 hours)</th>
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      </tr>
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    </thead>
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    <tfoot>
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    <tbody>
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      <tr>
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        <th scope="row">Negative Control (Colony 1)</th>
 +
        <td data-title="Abs600">0,461</td>
 +
        <td data-title="OD600 before">1,435</td>
 +
        <td data-title="Volume (ml) added to 12 ml media" data-type="currency">0,167</td>
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        <td data-title="OD600 after dilution (0 hours)" data-type="currency">0,06483974</td>
 +
      </tr>
 +
      <tr>
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        <th scope="row">Negative Control (Colony 2)</th>
 +
        <td data-title="Abs600">0,463</td>
 +
        <td data-title="OD600 before">1,439</td>
 +
        <td data-title="Volume (ml) added to 12 ml media" data-type="currency">0,167</td>
 +
        <td data-title="OD600 after dilution (0 hours)" data-type="currency">0,02584249</td>
 +
      </tr>
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        <tr>
 +
        <th scope="row">Positive Control (Colony 1)</th>
 +
        <td data-title="Abs<sub>600</sub>">0,450</td>
 +
        <td data-title="OD<sub>600</sub> before">1,400</td>
 +
        <td data-title="Volume (ml) added to 12 ml media" data-type="currency">0,171</td>
 +
        <td data-title="OD<sub>600</sub> after dilution (0 hours)" data-type="currency">0,04467949</td>
 +
      </tr>
 +
    <tr>
 +
        <th scope="row">Positive Control (Colony 2)</th>
 +
        <td data-title="Abs<sub>600</sub>">0,474</td>
 +
        <td data-title="OD<sub>600</sub> before">1,475</td>
 +
        <td data-title="Volume (ml) added to 12 ml media" data-type="currency">0,163</td>
 +
        <td data-title="OD<sub>600</sub> after dilution (0 hours)" data-type="currency">0,01689103</td>
 +
      </tr>
 +
          <tr>
 +
        <th scope="row">Test Device 1: J23101+I13504 (Colony 1)</th>
 +
        <td data-title="Abs<sub>600</sub>">0,462</td>
 +
        <td data-title="OD<sub>600</sub> before">1,437</td>
 +
        <td data-title="Volume (ml) added to 12 ml media" data-type="currency">0,167</td>
 +
        <td data-title="OD<sub>600</sub> after dilution (0 hours)" data-type="currency">0,03043498</td>
 +
      </tr>
 +
        <tr>
 +
        <th scope="row">Test Device 1: J23101+I13504 (Colony 2)</th>
 +
        <td data-title="Abs<sub>600</sub>">0,472</td>
 +
        <td data-title="OD<sub>600</sub> before">1,469</td>
 +
        <td data-title="Volume (ml) added to 12 ml media" data-type="currency">0,163</td>
 +
        <td data-title="OD<sub>600</sub> after dilution (0 hours)" data-type="currency">0,01977106</td>
 +
      </tr>
 +
        <tr>
 +
        <th scope="row">Test Device 2: J23106+I13504 (Colony 1)</th>
 +
        <td data-title="Abs<sub>600</sub>">0,477</td>
 +
        <td data-title="OD<sub>600</sub> before">1,483</td>
 +
        <td data-title="Volume (ml) added to 12 ml media" data-type="currency">0,162</td>
 +
        <td data-title="OD<sub>600</sub> after dilution (0 hours)" data-type="currency">0,03175824</td>
 +
      </tr>
 +
        <tr>
 +
        <th scope="row">Test Device 2: J23106+I13504 (Colony 2)</th>
 +
        <td data-title="Abs<sub>600</sub>">0,454</td>
 +
        <td data-title="OD<sub>600</sub> before">1,410</td>
 +
        <td data-title="Volume (ml) added to 12 ml media" data-type="currency">0,170</td>
 +
        <td data-title="OD<sub>600</sub> after dilution (0 hours)" data-type="currency">0,018837</td>
 +
      </tr>
 +
          <tr>
 +
        <th scope="row">Test Device 3: J23117+I13504 (Colony 1)</th>
 +
        <td data-title="Abs<sub>600</sub>">0,507</td>
 +
        <td data-title="OD<sub>600</sub> before">1,577</td>
 +
        <td data-title="Volume (ml) added to 12 ml media" data-type="currency">0,152</td>
 +
        <td data-title="OD<sub>600</sub> after dilution (0 hours)" data-type="currency">0,03261447</td>
 +
      </tr>
 +
          <tr>
 +
        <th scope="row">Test Device 3: J23117+I13504 (Colony 2)</th>
 +
        <td data-title="Abs<sub>600</sub>">0,492</td>
 +
        <td data-title="OD<sub>600</sub> before">1,529</td>
 +
        <td data-title="Volume (ml) added to 12 ml media" data-type="currency">0,157</td>
 +
        <td data-title="OD<sub>600</sub> after dilution (0 hours)" data-type="currency">0,01471154</td>
 +
      </tr>
 +
          <tr>
 +
        <th scope="row">Test Device 4: J23101.BCD2.E0040.B0015 (Colony 1)</th>
 +
        <td data-title="Abs<sub>600</sub>">0,420</td>
 +
        <td data-title="OD<sub>600</sub> before">1,307</td>
 +
        <td data-title="Volume (ml) added to 12 ml media" data-type="currency">0,184</td>
 +
        <td data-title="OD<sub>600</sub> after dilution (0 hours)" data-type="currency">0,03759615</td>
 +
      </tr>
 +
          <tr>
 +
        <th scope="row">Test Device 4: J23101.BCD2.E0040.B0015 (Colony 2)</th>
 +
        <td data-title="Abs600">0,472</td>
 +
        <td data-title="OD600 before">1,468</td>
 +
        <td data-title="Volume (ml) added to 12 ml media" data-type="currency">0,163</td>
 +
        <td data-title="OD600 after dilution (0 hours)" data-type="currency">0,01860348</td>
 +
      </tr>
 +
      <tr>
 +
        <th scope="row">Test Device 5: J23106.BCD2.E0040.B0015 (Colony 1)</th>
 +
        <td data-title="Abs600">0,513</td>
 +
        <td data-title="OD600 before">1,595</td>
 +
        <td data-title="Volume (ml) added to 12 ml media" data-type="currency">0,150</td>
 +
        <td data-title="OD600 after dilution (0 hours)" data-type="currency">0,04234432</td>
 +
      </tr>
 +
      <tr>
 +
        <th scope="row">Test Device 5: J23106.BCD2.E0040.B0015 (Colony 2)</th>
 +
        <td data-title="Abs600">0,467</td>
 +
        <td data-title="OD600 before">1,451</td>
 +
        <td data-title="Volume (ml) added to 12 ml media" data-type="currency">0,165</td>
 +
        <td data-title="OD600 after dilution (0 hours)" data-type="currency">0,01494505</td>
 +
      </tr>
 +
      <tr>
 +
        <th scope="row">Test Device 6: J23117.BCD2.E0040.B0015 (Colony 1)</th>
 +
        <td data-title="Abs600">0,482</td>
 +
        <td data-title="OD600 before">1,498</td>
 +
        <td data-title="Volume (ml) added to 12 ml media" data-type="currency">0,160</td>
 +
        <td data-title="OD600 after dilution (0 hours)" data-type="currency">0,04452381</td>
 +
      </tr>
 +
      <tr>
 +
        <th scope="row">Test Device 6: J23117.BCD2.E0040.B0015 (Colony 2)</th>
 +
        <td data-title="Abs600">0,455</td>
 +
        <td data-title="OD600 before">1,416</td>
 +
        <td data-title="Volume (ml) added to 12 ml media" data-type="currency">0,170</td>
 +
        <td data-title="OD600 after dilution (0 hours)" data-type="currency">0,02008242</td>
 +
      </tr>
 +
    </tbody>
 +
  </table>
 +
</div>
  
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<p>We have identified three mechanisms we believe to be mandatory for the development of a stable endosymbiotic relationship, which we will be trying to replicate in free-living cells. First of all, in order for the relationship to be stable, the two organisms must  be mutually dependent on each other; there must be a mutually beneficial interaction between host and symbiont. Secondly, there has to be some sort of control and synchronization of symbiont replication. If the symbiont were to be replicating freely we could end up with way too many or not enough symbionts in the host.  Finally, a common feature of the endosymbiotic organelles we have looked at, is the transfer of genes from the symbiont to the host. Because of this transfer, the gene and protein expression is taking place in the nucleus and the proteins and metabolites are transported to the organelle. This import of proteins is interesting not just for understanding endosymbiosis, but also for the potential applications in synthetic biology.</p>
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<p>Based on these considerations, we decided to work on three distinct, but intertwined, projects pertaining to endosymbiosis, namely Interdependence, Number Control, and Protein import. We believe that by combining these three projects, a key step towards the understanding of endosymbiosis and its employment in synthetic biology will be obtained. </p>
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                    <h2 class="section-heading">Applications and Implications</h2>
 
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                    <<p>By understanding the basic principles behind the creation of stable endosymbiotic events we hope that in the future it will be possible to use artificial endosymbiosis as a new technology in synthetic biology, and we believe that value can be created in the foundational track of the iGEM competition. History has shown that great scientific advances has followed the implementation of new revolutionary technologies (Gershon 2003). </p>
 
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<p>We envision that artificial endosymbiosis could be applied in a broad range of fields, including agriculture, medicine and production of valuable compounds. A deeper understanding of the relationships intertwining endosymbionts and their hosts could unravel new knowledge applicable for the treatment of mitochondrial diseases, while a living compartment able to fixate nitrogen from the air could decrease the fertilizer use in agricultural production. </p>
 
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<p>However, the applications are only limited by the imagination of future users. Indeed, the game-changing role of endosymbiosis has not gone unseen to the eyes of the modern bioengineers, who predict that the establishment of a novel interaction has the potential to radically alter the host cell physiology without directly affecting the host genome (Scientific America Vol 105 pp. 36-45).</p>
 
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<p>Before the potential application of artificial endosymbiosis, there are many things to consider. While the current regulations regarding GMO limits what is possible to apply in agriculture and medicine, regulations regarding synthetically modified organisms (SMOs) have not yet been systematically put into place. How will a new field of SMO be regulated, and how will it influence possible applications of artificial endosymbiosis?</p>
 
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<p>In addition to our scientific investigation we are enthused to trigger debate about synthetic biology. We intend to podcast intriguing conversations with experts, thereby hoping to reach the general public and impel the discussion about the ethics and future prospects in combining biology and engineering.</p>
 
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                     <h2>Find Incell here:</h2>
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Revision as of 11:13, 31 October 2017

I N T E R L A B

Cell measurements

Two colonies from each transformation were picked, and grown in foil-covered 50 ml falcon tubes over night (18 hours).

Preparation OD was measured, and a dilution was calculated to achieve an OD of 0.02. Dilution calculations can be found in the table next to this. Here we used the calculated correction factor from our initial abs/OD calibration. From the absorption measurements taken at 0 hours, we see indications of pipetting errors, as the OD600 (average) ranges from 0.015 to 0.6 (table 1).

Find Incell here: