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| − | <li><a href="https://2016.igem.org/Team:Edinburgh_UG/Team">Team</a></li>
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| − | <li><a href="https://2016.igem.org/Team:Edinburgh_UG/Description">Project</a></li>
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| − | <li><a href="https://2016.igem.org/Team:Edinburgh_UG/Description">Description</a></li>
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| − | <li><a href="https://2016.igem.org/Team:Edinburgh_UG/Proof">Proof of Concept</a></li>
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| − | <li><a href="https://2016.igem.org/Team:Edinburgh_UG/Demonstrate">Demonstrate</a></li>
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| − | <li><a href="https://2016.igem.org/Team:Edinburgh_UG/Results">Results</a></li>
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| − | <li><a href="https://2016.igem.org/Team:Edinburgh_UG/Notebook">Notebook</a></li>
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| − | </ul>
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| − | <li><a href="https://2016.igem.org/Team:Edinburgh_UG/Plate_Reader">Plate Reader</a></li>
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| − | <h2 class="section-heading">Introduction</h2>
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| − | The term InterLab stands for Inter-Laboratory studies. The iGEM InterLab studies conducted duringthe last two years have been of huge importance to establish a standard for reproducibility offluorescent measurements. The measurement of fluorescence is an essential tool in synthetic biology.However, the absence of absolute units and standard protocols makes it harder to interpret data and share biological parts between laboratories. The 2016 iGEM InterLab consists of two protocols formeasuring GFP that will lead to the creation of absolute units. This collection of measurements can then be used to establish the accuracy with which GFP is measured around the world and what needs to be improved when following a standard protocol. As a team, we decided that the iGEM InterLab was a great opportunity to improve our laboratory skills and take part into an international study.
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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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| − | <p>The protocol supplied in the iGEM 2015 InterLab Protocol form was followed with a few minor changes.
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| − | The aim of the 2016 iGEM InterLab was to quantify the expression of GFP under different promoters and Ribosomal Binding Regions (RBRs). The use of GFP to indirectly measure the expression of a specific protein in cells is a key approach in biology. It enables us to monitor expression levels without killing the cells. The ability measure expression levels per cell is important to be able to quantify strength of promotors. In order to achieve this, population size is indirectly measured using OD600. Expression per cell is then obtained by Fluorescence/OD600.</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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| − | <p>The cells were grown on LB agar supplemented with 1000x chloramphenicol.</p>
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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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| − | <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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| − | <p>A SpectaMax M5 plate reader was used to obtain the values of fluorescence.</p>
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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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| − | <p>The data is in relative fluorescent units.</p>
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| − | <p>1.) 1278.712 <br> 2.) 1216.887 <br> 3.) 1110.834</p>
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| − | <p>1.) -36.863 <br> 2.) -33.066 <br> 3.) -44.696</p>
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| − | <p>1.)5082.078 <br> 2.) 5879.239 <br> 3.) 5973.973
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| − | Mean = 5645.097
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| − | Standard Deviation = 489.884
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| − | 1.) 3185.188 <br> 2.) 3119.913 <br> 3.) 2396.115
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| − | Mean = 2900.405
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| − | 1.) -14.003 <br> 2.) -27.149 <br> 3.) -16.631
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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>
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| − | </h4>
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| − | <div id="collapseFourteen" class="panel-collapse collapse">
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| − | <p>2nd Replicate = 5202.079 <br> 3rd replicate = 5187.619</p>
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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>
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| − | </h4>
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| − | <div id="collapseFifteen" class="panel-collapse collapse">
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| − | <p>2nd replicate = 6008.646 <br> 3rd replicate = 6135.483</p>
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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>
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| − | </h4>
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| − | </div>
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| − | <div id="collapseSixteen" class="panel-collapse collapse">
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| − | <p>2nd replicate =6634.865 <br> 3rd replicate = 6254.715</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="#collapseSeventeen">Mean and standard deviation of technical replicates of J23101 + I13504 (device 1)</a>
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| − | </h4>
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| − | <div id="collapseSeventeen" class="panel-collapse collapse">
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| − | <p>
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| − | Mean = 5817.633
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| − | Standard deviation = 541.009
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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>
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| − | </h4>
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| − | </div>
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| − | <div id="collapseEighteen" class="panel-collapse collapse">
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| − | <p>2nd replicate = 3694.877 <br> 3rd replicate = 3606.107</p>
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| − | </div>
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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>
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| − | </h4>
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| − | </div>
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| − | <div id="collapseNineteen" class="panel-collapse collapse">
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| − | <div class="panel-body">
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| − | <p>2nd replicate = 3036.241 <br> 3rd replicate = 3140.1</p>
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| − | </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="#collapseTwenty">Fluorescence of 2nd and 3rd technical replicates of J23106 + I13504 (device 2) biological replicate 3</a>
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| − |
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| − | </h4>
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| − | </div>
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| − | <div id="collapseTwenty" class="panel-collapse collapse">
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| − | <div class="panel-body">
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| − | <p>2nd replicate = 2871.642 <br> 3rd replicate = 2801.428</p>
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <div class="panel panel-default">
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| − |
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| − | <div class="panel-heading">
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| − |
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| − | <h4 class="panel-title">
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| − |
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| − | <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwentyOne">Mean and standard deviation of technical replicates of J23106 + I13504 (device 2)</a>
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| − |
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| − | </h4>
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| − | </div>
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| − | <div id="collapseTwentyOne" class="panel-collapse collapse">
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| − | <div class="panel-body">
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| − |
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| − | <p>Mean = 3094.623
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| − | <br>
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| − | Standard deviation = 396.837</p>
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| − |
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| − | </div>
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| − | </div>
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| − |
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| − | </div>
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| − |
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| − | <div class="panel panel-default">
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| − | <div class="panel-heading">
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| − |
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| − | <h4 class="panel-title">
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| − |
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| − | <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>
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| − |
| |
| − | </h4>
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| − |
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| − | </div>
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| − |
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| − | <div id="collapseTwentyTwo" class="panel-collapse collapse">
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| − | <div class="panel-body">
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| − |
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| − | <p>2nd replicate = 7.859 <br> 3rd replicate = 0.659</p>
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| − |
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| − | </div>
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| − |
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| − | </div>
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| − |
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| − | </div>
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| − |
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| − | <div class="panel panel-default">
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| − |
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| − | <div class="panel-heading">
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| − |
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| − | <h4 class="panel-title">
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| − |
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| − | <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>
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| − |
| |
| − | </h4>
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| − |
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| − | </div>
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| − |
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| − | <div id="collapseTwentyThree" class="panel-collapse collapse">
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| − |
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| − | <div class="panel-body">
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| − |
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| − | <p>2nd replicate = 28.499 <br> 3rd replicate = 11.4</p>
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| − |
| |
| − | </div>
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| − |
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| − | </div>
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| − |
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| − | </div>
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| − |
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| − | <div class="panel panel-default">
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| − |
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| − | <div class="panel-heading">
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| − |
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| − | <h4 class="panel-title">
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| − |
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| − | <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>
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| − |
| |
| − | </h4>
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| − |
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| − | </div>
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| − |
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| − | <div id="collapseTwentyFour" class="panel-collapse collapse">
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| − |
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| − | <div class="panel-body">
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| − |
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| − | <p>2nd replicate = -17.823 <br> 3rd replicate = -6.295</p>
| |
| − |
| |
| − | </div>
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| − |
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| − | </div>
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| − |
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| − | </div>
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| − |
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| − | <div class="panel panel-default">
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| − |
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| − | <div class="panel-heading">
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| − |
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| − | <h4 class="panel-title">
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| − |
| |
| − | <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwentyFive">Mean and standard deviation of technical replicates of J23117 + I13504 (device 3)</a>
| |
| − |
| |
| − | </h4>
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| − |
| |
| − | </div>
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| − |
| |
| − | <div id="collapseTwentyFive" class="panel-collapse collapse">
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| − |
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| − | <div class="panel-body">
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| − |
| |
| − | <p>Mean = -3.720
| |
| − | <br>
| |
| − | Standard deviation = 17.489</p>
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| − |
| |
| − | </div>
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| − |
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| − | </div>
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| − |
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| − | </div>
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| − | </div>
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| − | </div>
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| − | <footer>
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| − | <p class="pull-right"><a href="#">Back to top</a></p>
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