Difference between revisions of "Team:Edinburgh/InterLab"

 
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/InterLab">InterLab</a></li>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Notebook/PMADetection">PMA Detection</a> </li>
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       <header class="intro">
 
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                 <h1 class="brand-heading">InterLab Study</h1>
 
                 <h1 class="brand-heading">InterLab Study</h1>
 
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                        <span class="arrowtext">Scroll down to read more</span>
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                     <h2 class="section-heading">Overview</h2>
 
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                      The Edinburgh iGEM 2015 team is participating in the second year of the iGEM InterLab study along with 82 other teams. The 2015 InterLab study focuses on obtaining consistent fluorescence data for three genetic devices expressing GFP. The devices consist of a constitutive promoter with either high (J23101), medium (J23106) or low (J23117) promoter strengths, fused to GFP (I13504).
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The promoters and GFP from the Biobrick distribution kit were transformed into E.coli Top10 cells then digested to have complementary sticky ends and ligated resulting in the final devices. The devices were validated by restriction digest and sequence data. Each promoter fused to GFP was transformed once more grown in cultures overnight and then measurements of fluorescence were taken.
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More detailed information regarding the cloning process can be found below and on our InterLab lab notebook page.
 
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                      All other protocols used in the process of the study can be found on the <a href="2015.igem.org/Team:Edinburgh/Project/Protocols">protocols</a> page.
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseOne">What protocol was followed for growing cells for measurement?</a>
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                    <p>The protocol supplied in the iGEM 2015 InterLab Protocol  form was followed with a few minor changes.
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                    Each device and the two controls were streaked out onto LB agar plates supplemented with chloramphenicol and incubated at 37°C for 22 hours.  3 colonies from each plate were inoculated into 50ml Falcon tubes containing 10ml of LB media and chloramphenicol.  The tubes were incubated upright on a platform shaker in a room kept at 37°C for 16 hours.</p>
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwo">What controls were used?</a>
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                    <p>The positive control used was the recommended BBa_I20270, a chloramphenicol resistant GFP expression device in pSB1C3.
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The negative control used consisted of cells transformed with BioBrick number BBa_R0040. This part consists of the promoter pTetR and should therefore have very low fluorescence</p>
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseThree">What agar was used to grow the cells?</a>
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                    <p>The cells were grown on LB agar supplemented with 1000x chloramphenicol.</p>
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseFour">How were the cells prepared for measurement?</a>
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                    <p>A colony of cells was inoculated into 10ml of liquid LB with 10µl of 1000x chloramphenicol. The cultures were grown in a 50ml falcon tube, upright on shaking platform at 275rpm in 37°C room overnight.
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For each device three colonies were inoculated to act as the three biological replicates.</p>
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseFive">How was the fluorescence of each device measured? (followed protocol)</a>
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                    <p>After the overnight incubation period of 16 hours the liquid cultures were removed from the 37°C room. 200µl from each culture was added to a 96-well plate. The initial optical density of each sample was measured at 600nm using a plate reader. All samples were then diluted appropriately so as to have an OD600 value within 5% of 0.5.  Two additional technical replicates for each of the biological replicates were set up at this point. The OD600 was measured again to ensure all samples had an optical density within 5% of 0.5 before the fluorescence measurements were taken.
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To measure fluorescence, the cells were excited at a wavelength of 485nm and emitted light at 538nm as measured.
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The values obtained for fluorescence were in relative fluorescence units.</p>
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseSix">What instrument was used to measure the fluorescence of each device?</a>
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                    <p>A SpectaMax M5 plate reader was used to obtain the values of fluorescence.</p>
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                <div class="col-lg-12 text-center">
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                    <h2 class="section-heading">Results</h2>                   
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                </div>
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            </div>
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  </div>
 
</section>
 
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            <div class="panel-heading">
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                <h4 class="panel-title">
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseSeven">How was the raw data processed?</a>
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                </h4>
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            <div id="collapseSeven" class="panel-collapse collapse">
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                <div class="panel-body">
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                    <p>The background absorbance of 6 wells containing 200µl of LB was measured.  The average of these readings was then subtracted from the fluorescence values obtained for the devices.</p>
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 +
                </div>
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            <div class="panel-heading">
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                <h4 class="panel-title">
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseEight">What were the units of the results?</a>
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                </h4>
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            </div>
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            <div id="collapseEight" class="panel-collapse collapse">
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                <div class="panel-body">
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                    <p>The data is in relative fluorescent units.</p>
 +
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            <div class="panel-heading">
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                <h4 class="panel-title">
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseNine">Fluorescence of 3 biological replicates of positive control</a>
 +
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                </h4>
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            </div>
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            <div id="collapseNine" class="panel-collapse collapse">
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                <div class="panel-body">
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<p>1.) 1278.712 <br>  2.) 1216.887  <br>  3.) 1110.834</p>
 +
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                </div>
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            <div class="panel-heading">
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                <h4 class="panel-title">
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTen">Fluorescence of 3 biological replicates of negative control</a>
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                </h4>
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            <div id="collapseTen" class="panel-collapse collapse">
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                <div class="panel-body">
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                    <p>1.) -36.863 <br>  2.) -33.066 <br>    3.) -44.696</p>
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                <h4 class="panel-title">
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseEleven">Fluorescence of 3 biological replicates of J23101 + I13504, mean and st.dev (device 1)</a>
 +
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                </h4>
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            </div>
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            <div id="collapseEleven" class="panel-collapse collapse">
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                <div class="panel-body">
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                    <p>1.)5082.078 <br> 2.) 5879.239 <br> 3.) 5973.973
 +
                        <br>
 +
                        Mean = 5645.097
 +
                        <br>
 +
                        Standard Deviation = 489.884
 +
                  </p>
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                <h4 class="panel-title">
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwelve">Fluorescence of 3 biological replicates of J23106 + I13504, mean and st.dev (device 2)</a>
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                </h4>
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            <div id="collapseTwelve" class="panel-collapse collapse">
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                <div class="panel-body">
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                    <p>
 +
                      1.) 3185.188 <br> 2.) 3119.913 <br> 3.) 2396.115
 +
                      <br>
 +
                      Mean = 2900.405
 +
                      <br>
 +
                      Standard Deviation = 437.946
 +
                  </p>
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          <div class="panel panel-default">
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            <div class="panel-heading">
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                <h4 class="panel-title">
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseThirteen">Fluorescence of 3 biological replicates of J23117 + I13504, mean and st.dev (device3)</a>
 +
 +
                </h4>
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            </div>
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            <div id="collapseThirteen" class="panel-collapse collapse">
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                <div class="panel-body">
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                    <p>
 +
                      1.) -14.003 <br> 2.) -27.149 <br> 3.) -16.631
 +
                      <br>
 +
                      Mean = -19.261
 +
                      <br>
 +
                      Standard Deviation = 6.956
 +
                  </p>
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                </div>
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            </div>
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        </div>
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    </div>
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</div>
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<section id="about">
 +
    <div class="container">
 +
            <div class="row">
 +
                <div class="col-lg-12 text-center">
 +
                    <h2 class="section-heading">Extra Credit</h2>                   
 +
                </div>
 +
            </div>
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  </div>
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</section>
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<div class="container">
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    <div id="accordion" class="panel-group">
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            <div class="panel-heading">
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                <h4 class="panel-title">
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseFourteen">Fluorescence of 2nd and 3rd technical replicates of J23101 + I13504 (device 1) biological replicate 1</a>
 +
 +
                </h4>
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            </div>
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            <div id="collapseFourteen" class="panel-collapse collapse">
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                <div class="panel-body">
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                    <p>2nd Replicate = 5202.079 <br> 3rd replicate = 5187.619</p>
 +
 +
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            <div class="panel-heading">
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                <h4 class="panel-title">
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseFifteen">Fluorescence of 2nd and 3rd technical replicates of J23101 + I13504 (device 1) biological replicate 2</a>
 +
 +
                </h4>
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            </div>
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            <div id="collapseFifteen" class="panel-collapse collapse">
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                <div class="panel-body">
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                    <p>2nd replicate = 6008.646 <br> 3rd replicate = 6135.483</p>
 +
 +
                </div>
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            </div>
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        </div>
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        <div class="panel panel-default">
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            <div class="panel-heading">
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                <h4 class="panel-title">
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseSixteen">Fluorescence of 2nd and 3rd technical replicates of J23101 + I13504 (device 1) biological replicate 3</a>
 +
 +
                </h4>
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            </div>
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            <div id="collapseSixteen" class="panel-collapse collapse">
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                <div class="panel-body">
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                    <p>2nd replicate =6634.865 <br> 3rd replicate = 6254.715</p>
 +
 +
                </div>
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            </div>
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        </div>
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        <div class="panel panel-default">
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            <div class="panel-heading">
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                <h4 class="panel-title">
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 +
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseSeventeen">Mean and standard deviation of technical replicates of J23101 + I13504 (device 1)</a>
 +
 +
                </h4>
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            </div>
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            <div id="collapseSeventeen" class="panel-collapse collapse">
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                <div class="panel-body">
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                    <p>
 +
Mean = 5817.633
 +
<br>
 +
Standard deviation = 541.009
 +
</p>
 +
 +
                </div>
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            </div>
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                <div class="panel panel-default">
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            <div class="panel-heading">
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                <h4 class="panel-title">
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseEighteen">Fluorescence of 2nd and 3rd technical replicates of J23106 + I13504 (device 2) biological replicate 1</a>
 +
 +
                </h4>
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            </div>
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            <div id="collapseEighteen" class="panel-collapse collapse">
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                <div class="panel-body">
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                    <p>2nd replicate = 3694.877 <br> 3rd replicate = 3606.107</p>
 +
 +
                </div>
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            </div>
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        </div>
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                <div class="panel panel-default">
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            <div class="panel-heading">
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                <h4 class="panel-title">
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 +
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseNineteen">Fluorescence of 2nd and 3rd technical replicates of J23106 + I13504 (device 2) biological replicate 2</a>
 +
 +
                </h4>
 +
 +
            </div>
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 +
            <div id="collapseNineteen" class="panel-collapse collapse">
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                <div class="panel-body">
 +
 +
                    <p>2nd replicate = 3036.241 <br> 3rd replicate = 3140.1</p>
 +
 +
                </div>
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            </div>
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        </div>
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    <div class="panel panel-default">
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            <div class="panel-heading">
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 +
                <h4 class="panel-title">
 +
 +
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwenty">Fluorescence of 2nd and 3rd technical replicates of J23106 + I13504 (device 2) biological replicate 3</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseTwenty" class="panel-collapse collapse">
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                <div class="panel-body">
 +
 +
                    <p>2nd replicate = 2871.642 <br> 3rd replicate = 2801.428</p>
 +
 +
                </div>
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            </div>
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        </div>
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    <div class="panel panel-default">
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            <div class="panel-heading">
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 +
                <h4 class="panel-title">
 +
 +
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwentyOne">Mean and standard deviation of technical replicates of J23106 + I13504 (device 2)</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseTwentyOne" class="panel-collapse collapse">
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 +
                <div class="panel-body">
 +
 +
                    <p>Mean = 3094.623
 +
<br>
 +
Standard deviation = 396.837</p>
 +
 +
                </div>
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            </div>
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 +
        </div>
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    <div class="panel panel-default">
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            <div class="panel-heading">
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                <h4 class="panel-title">
 +
 +
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwentyTwo">Fluorescence of 2nd and 3rd technical replicates of J23117 + I13504 (device 3) biological replicate 1</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseTwentyTwo" class="panel-collapse collapse">
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                <div class="panel-body">
 +
 +
                    <p>2nd replicate = 7.859 <br> 3rd replicate = 0.659</p>
 +
 +
                </div>
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 +
            </div>
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    <div class="panel panel-default">
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            <div class="panel-heading">
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 +
                <h4 class="panel-title">
 +
 +
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwentyThree">Fluorescence of 2nd and 3rd technical replicates of J23117 + I13504 (device 3) biological replicate 2</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseTwentyThree" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
 +
                    <p>2nd replicate = 28.499 <br> 3rd replicate = 11.4</p>
 +
 +
                </div>
 +
 +
            </div>
 +
 +
        </div>
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 +
    <div class="panel panel-default">
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            <div class="panel-heading">
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                <h4 class="panel-title">
 +
 +
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwentyFour">Fluorescence of 2nd and 3rd technical replicates of J23117 + I13504 (device 3) biological replicate 3</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseTwentyFour" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
 +
                    <p>2nd replicate = -17.823 <br> 3rd replicate = -6.295</p>
 +
 +
                </div>
 +
 +
            </div>
 +
 +
        </div>
 +
 +
    <div class="panel panel-default">
 +
 +
            <div class="panel-heading">
 +
 +
                <h4 class="panel-title">
 +
 +
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwentyFive">Mean and standard deviation of technical replicates of J23117 + I13504 (device 3)</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseTwentyFive" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
 +
                    <p>Mean = -3.720
 +
<br>
 +
Standard deviation = 17.489</p>
 +
 +
                </div>
 +
 +
            </div>
 +
 +
        </div>
 +
 +
    </div>
 +
</div>
 +
 +
  
 
       <footer>
 
       <footer>

Latest revision as of 18:58, 20 November 2015

Overview

The Edinburgh iGEM 2015 team is participating in the second year of the iGEM InterLab study along with 82 other teams. The 2015 InterLab study focuses on obtaining consistent fluorescence data for three genetic devices expressing GFP. The devices consist of a constitutive promoter with either high (J23101), medium (J23106) or low (J23117) promoter strengths, fused to GFP (I13504).

The promoters and GFP from the Biobrick distribution kit were transformed into E.coli Top10 cells then digested to have complementary sticky ends and ligated resulting in the final devices. The devices were validated by restriction digest and sequence data. Each promoter fused to GFP was transformed once more grown in cultures overnight and then measurements of fluorescence were taken.

More detailed information regarding the cloning process can be found below and on our InterLab lab notebook page.

Protocols

All other protocols used in the process of the study can be found on the protocols page.

The protocol supplied in the iGEM 2015 InterLab Protocol form was followed with a few minor changes.
Each device and the two controls were streaked out onto LB agar plates supplemented with chloramphenicol and incubated at 37°C for 22 hours. 3 colonies from each plate were inoculated into 50ml Falcon tubes containing 10ml of LB media and chloramphenicol. The tubes were incubated upright on a platform shaker in a room kept at 37°C for 16 hours.

The positive control used was the recommended BBa_I20270, a chloramphenicol resistant GFP expression device in pSB1C3.
The negative control used consisted of cells transformed with BioBrick number BBa_R0040. This part consists of the promoter pTetR and should therefore have very low fluorescence

The cells were grown on LB agar supplemented with 1000x chloramphenicol.

A colony of cells was inoculated into 10ml of liquid LB with 10µl of 1000x chloramphenicol. The cultures were grown in a 50ml falcon tube, upright on shaking platform at 275rpm in 37°C room overnight.
For each device three colonies were inoculated to act as the three biological replicates.

After the overnight incubation period of 16 hours the liquid cultures were removed from the 37°C room. 200µl from each culture was added to a 96-well plate. The initial optical density of each sample was measured at 600nm using a plate reader. All samples were then diluted appropriately so as to have an OD600 value within 5% of 0.5. Two additional technical replicates for each of the biological replicates were set up at this point. The OD600 was measured again to ensure all samples had an optical density within 5% of 0.5 before the fluorescence measurements were taken.
To measure fluorescence, the cells were excited at a wavelength of 485nm and emitted light at 538nm as measured.
The values obtained for fluorescence were in relative fluorescence units.

A SpectaMax M5 plate reader was used to obtain the values of fluorescence.

Results

The background absorbance of 6 wells containing 200µl of LB was measured. The average of these readings was then subtracted from the fluorescence values obtained for the devices.

The data is in relative fluorescent units.

1.) 1278.712
2.) 1216.887
3.) 1110.834

1.)5082.078
2.) 5879.239
3.) 5973.973
Mean = 5645.097
Standard Deviation = 489.884

1.) 3185.188
2.) 3119.913
3.) 2396.115
Mean = 2900.405
Standard Deviation = 437.946

1.) -14.003
2.) -27.149
3.) -16.631
Mean = -19.261
Standard Deviation = 6.956

Extra Credit