Difference between revisions of "Team:UCopenhagen/InterLab"

 
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                         <h1>I N T E R  L A B</h1>   
 
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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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                    <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/b/b9/InterLab-figure_UCopenhagen.png" alt="" width="250" height="200">
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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">Cell measurements </h2>
 +
                    <p class="lead">Two colonies from each transformation were picked, and grown in foil-covered 50 ml falcon tubes over night (18 hours). </p>
 +
<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>
 +
      <tr>
 +
<th scope="col"></th>
 +
        <th scope="col">Abs<sub>600</sub></th>
 +
        <th scope="col">OD<sub>600</sub> before</th>
 +
        <th scope="col">Volume (ml) added to 12 ml media</th>
 +
        <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>
 +
    </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>
 +
        <td data-title="OD600 after dilution (0 hours)" data-type="currency">0,06483974</td>
 +
      </tr>
 +
      <tr>
 +
        <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>
 +
        <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>
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</div>
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<p class="lead"><strong>Natural endosymbiosis (our model)</strong> Shortly: what is endosymbiosis?
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<strong> Synthetic endosymbiosis </strong> will have innumerable applications if developed to be the orthogonal system we envision. We have chosen to take a bottom-up approach to it, through the investigation of the following three essential mechanisms.  
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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.
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Add something about how our idea started?
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<figure>
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                    <img class="img-responsive3" src="https://static.igem.org/mediawiki/2017/3/37/Interlab_OD_positive.png" alt="" width="250" height="200">
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<strong> Interdependence </strong> between host and endosymbiont is necessary in order for the endosymbiotic relationship to be stable and maintained throughout evolution or, more relevant for synthetic biology, through generations. The relationship should be beneficial and crucial for the host and the endosymbiont alike. Interdependency would thus decrease possible safety concerns: without the dependency relationship, the endosymbiotic relationship will not spread and be maintained in a wild population.
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<figcaption><b>Figure 2 </b>Fluorescence measurement in high-fluorescing samples (positive control and test devices 1, 2 and 4) over 6 hours.
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<strong>Number control</strong> addresses our concern that the endosymbiont will thrive too well in the host, and replicate uncontrollably - thus overwhelming the host system - or be lost in host replications. Thus we strive for a way to maintain a stable number of endosymbionts in the system. A control system that repress endosymbiont reproduction in high concentration/numbers, and allow replication when the concentration is decreased, would address this concern.
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                    <img class="img-responsive3" src="https://static.igem.org/mediawiki/2017/d/d8/Interlab_OD_negative.png" alt="" width="250" height="200">
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<br>
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<figcaption><b>Figure 3 </b>OD measurements in low-fluorescing samples (negative control and test devides 3, 5 and 6) over 6 hours.
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                    <h2 class="section-heading">Fluorescence</h2>
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 +
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.  
 +
<figure>
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                    <img class="img-responsive3" src="https://static.igem.org/mediawiki/2017/d/df/Interlab_fluorescence_positive.png" alt="" width="250" height="200">
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<figcaption><b>Figure 4 </b>Fluorescence measurement in high-fluorescing samples (positive control and test devices 1, 2 and 4) over 6 hours.
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                    <img class="img-responsive3" src="https://static.igem.org/mediawiki/2017/e/e4/Interlab_fluorescence_negative.png" alt="" width="250" height="200">
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<figcaption><b>Figure 5 </b>Fluorescence measurements in low-fluorescing samples (negative control and test devides 3, 5 and 6) over 6 hours.
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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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<strong>Protein import</strong> is a common trait observed in naturally occurring endosymbiotic relationships, such as mitochondria and chloroplasts. In these cases a down regulation of protein expression in the symbiont is observed, and the protein expression is instead moved to the cell. Proteins expressed in mitochondria and chloroplasts are transcribed in the nucleus, translated in the cytoplasm and subsequently transported across the cell membranes into the endosymbionts. This mechanism would tighten the relationship between host and symbiont, and would allow endosymbiotic manipulation of precursors produced in the host. </p>
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Latest revision as of 20:10, 1 November 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.


Figure 2 Fluorescence measurement in high-fluorescing samples (positive control and test devices 1, 2 and 4) over 6 hours.


Figure 3 OD measurements in low-fluorescing samples (negative control and test devides 3, 5 and 6) over 6 hours.

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.


Figure 4 Fluorescence measurement in high-fluorescing samples (positive control and test devices 1, 2 and 4) over 6 hours.


Figure 5 Fluorescence measurements in low-fluorescing samples (negative control and test devides 3, 5 and 6) over 6 hours.

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.

Find Incell here: