Difference between revisions of "Team:UCopenhagen/Collaborations"

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                         <h1>C O L L A B O R A T I O N S</h1>
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                     <h2 class="section-heading">Introduction </h2>
 
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                     <p class="lead">We participated in InterLitab, as we want to be contribute to the scientific progress made through this globe spanning project. In InterLab, 6 test devices are inserted in <i>E.coli</i> D5 α, and the growth and fluorescence is measured. <br><br>
  
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Collaboration is a powerful driver of progress in science. To stand on the shoulders of giants is increasingly unrealistic in a globalised world where scientists work in ever more specialised areas and without geographic or language barriers.
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We used the following plasmids provided by iGEM HQ to transform <i>E.coli</i>:
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For science to continue to benefit from globalisation, we must advance the axiom and seek to build a human pyramid from the shoulders before and around us. Collaboration and open source data are at the heart of iGEM and so too the heart of Incell.  
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<li>Positive control</li>
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<li>Negative control</li>
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Below we describe our most significant collaborations within the iGEM community. The symbiosis we strive for in our project has shone brightly through the partnerships established with teams around the world. Our story — and the stories of our friends — have been mutually shaped by the diversity of our shared experiences, knowledge and perspectives.
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<li>Test Device 1: J23101+I13504 </li>
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<br><br>
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<li>Test Device 2: J23106+I13504 </li>
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</p>
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<li>Test Device 3: J23117+I13504  </li>
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<li>Test Device 4: J23101.BCD2.E0040.B0015 </li>
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<li>Test Device 5: J23106.BCD2.E0040.B0015 </li>
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<li>Test Device 6: J23117.BCD2.E0040.B0015 </li>
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                    <h2 class="section-heading">Calibrations</h2>
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                    <p class="lead"> Before our measurements began, we performed some calibrations: First an OD<sub>600</sub> reference point for our plate reader, performed with LUDOX according to the protocol. Here we found a correction factor which can be used to calculate OD from measured absorbance. Our correction fator is 3.11. <br><br>
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Secondly we made a fluorescence standard curve with a serial dilution of fluorescein (figure 1). We used the lower 5 data points to calculate a mean µM fluorescein pr a.u. We chose to use the lower concentration range due to two factors: 1) Linearity is better for the lower fluorescein concentrations, and 2) our measured data has a maximum fluorescence of 500, which makes it more important to have a good fit in the lower range.
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<figcaption><b>Figure 1 </b>Standard curve of fluorescein fluorescence.
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Fluorescence in arbitraty units (a.u.), fluorescein concentration in µM.</figcaption>
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                     <h2 class="section-heading">Applications and Implications</h2>
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                     <h2 class="section-heading">Cell measurements </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 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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<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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    <caption><b>Table 1</b> Starting OD.</caption>
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    <thead>
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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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        <th scope="row">Negative Control (Colony 1)</th>
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        <td data-title="Abs600">0,461</td>
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        <td data-title="OD600 before">1,435</td>
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        <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>
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        <th scope="row">Negative Control (Colony 2)</th>
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        <td data-title="Abs600">0,463</td>
 +
        <td data-title="OD600 before">1,439</td>
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        <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">0,02584249</td>
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        <th scope="row">Positive Control (Colony 1)</th>
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        <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>
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        <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>
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        <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>
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      </tr>
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        <tr>
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        <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>
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      </tr>
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        <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>
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      </tr>
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        <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>
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        <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>
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        <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>
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        <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>
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        <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>
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      </tr>
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        <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>
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        <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>
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        <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>
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        <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>
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                    <h2 class="section-heading">OD<sub>600</sub </h2>
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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 class="lead">Cell growth stagnated between 4 and 6 hours. Cells transformed with Test Device 1 and 4 grew slower than the 6 other transformations, and even decreased in OD between 4 and 6 hours. Click on figures for enlarged images:</p>
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                    <h2 class="section-heading">Fluorescence</h2>
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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 class="lead">Some transformed cells continue to increase fluorescence despite a decrease in OD in the same samples. Devices 3 and 6 are very close to the negative control in fluorescence. Click on figures for enlarged images:</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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    <img class="fblogo" border="0" alt="Graph3" src="https://static.igem.org/mediawiki/2017/e/e5/Graph3_InterLab.png"/></a>
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                    <h2 class="section-heading">Conclusion </h2>
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                    <p class="lead">Our data indicate which plasmid elements induce the highest production of GFP.
 +
Plasmids containing J23117 (Test devices 3 and 6) does not express fluorescence to a higher degree than the negative control. Plasmids with J23101 (Device 1 and 4) induced the highest fluorescence, and plasmids containing J23106 (Test devices 2 and 5) were somewhere in between. Combining the J23101 or J23106 with I13504 (Test devices 1-3) gave a higher fluorescence than adding BCD2.E0040.B0015 (Test devices 4-6).
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These results are not reliable on their own, but will be more robust and reliable when combined with data from the other teams participating in the interlab study.
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Revision as of 11:31, 31 October 2017

I N T E R L A B

Introduction

We participated in InterLitab, as we want to be contribute to the scientific progress made through this globe spanning project. In InterLab, 6 test devices are inserted in E.coli D5 α, and the growth and fluorescence is measured.

We used the following plasmids provided by iGEM HQ to transform E.coli:

  • Positive control
  • Negative control
  • Test Device 1: J23101+I13504
  • Test Device 2: J23106+I13504
  • Test Device 3: J23117+I13504
  • Test Device 4: J23101.BCD2.E0040.B0015
  • Test Device 5: J23106.BCD2.E0040.B0015
  • Test Device 6: J23117.BCD2.E0040.B0015

Calibrations

Before our measurements began, we performed some calibrations: First an OD600 reference point for our plate reader, performed with LUDOX according to the protocol. Here we found a correction factor which can be used to calculate OD from measured absorbance. Our correction fator is 3.11.

Secondly we made a fluorescence standard curve with a serial dilution of fluorescein (figure 1). We used the lower 5 data points to calculate a mean µM fluorescein pr a.u. We chose to use the lower concentration range due to two factors: 1) Linearity is better for the lower fluorescein concentrations, and 2) our measured data has a maximum fluorescence of 500, which makes it more important to have a good fit in the lower range.



Figure 1 Standard curve of fluorescein fluorescence. Fluorescence in arbitraty units (a.u.), fluorescein concentration in µM.

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).

OD600

Cell growth stagnated between 4 and 6 hours. Cells transformed with Test Device 1 and 4 grew slower than the 6 other transformations, and even decreased in OD between 4 and 6 hours. Click on figures for enlarged images:

​

Fluorescence


Some transformed cells continue to increase fluorescence despite a decrease in OD in the same samples. Devices 3 and 6 are very close to the negative control in fluorescence. Click on figures for enlarged images:

​

Conclusion

Our data indicate which plasmid elements induce the highest production of GFP. Plasmids containing J23117 (Test devices 3 and 6) does not express fluorescence to a higher degree than the negative control. Plasmids with J23101 (Device 1 and 4) induced the highest fluorescence, and plasmids containing J23106 (Test devices 2 and 5) were somewhere in between. Combining the J23101 or J23106 with I13504 (Test devices 1-3) gave a higher fluorescence than adding BCD2.E0040.B0015 (Test devices 4-6).

These results are not reliable on their own, but will be more robust and reliable when combined with data from the other teams participating in the interlab study.

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