Difference between revisions of "Team:Exeter/Interlab"

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<a href="#section_1" class="banner_link col-xs-6 col-sm-3"><span class="oneline">Introduction and Aims</span></a>
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<a href="#section_2" class="banner_link col-xs-6 col-sm-3"><span class="oneline">Protocol and Results</span></a>
 
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Section 1
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Introduction and Aims
 
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<p id="pp">Interlab study for 2016 iGEM aimed to quantify expression of five different
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reporter constructs which have GFP under the control of differing promoters and ribosome
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binding sequences. This involved measuring the fluorescence/OD600 of the total population
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of DH5α E.coli cells with each construct to give an adjustment for relative expression per cell.
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The objective is to convert the relative unit of fluorescence to an absolute unit by calibrating
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each team’s instruments and comparing this to a measured standard curve.<p>
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Section 2
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Protocol and Results
 
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<p id="pp">Our team began on day 1 by measuring the standard LUDOX Abs600. This involved pipetting LUDOX and water into two separate columns of our 96 well plate and measuring the absorbance of the 4 replicates at 600 nm and standard Tecan mode.</p>
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<p id="pp">Later the same day we prepared a serial dilution of FITC provided in the interkab kit using PBS. This was pipetted into a 96 well plate and the fluorescence of all samples were measured in standard measurement modes. We then repeated these measurements to produce a series of standard curves. The fluorescence readings were taken at around 26.4°C at 477nm to 515nm excitation and emission respectively. We measured at a gain setting of 46,56,66. We found that 10 makes a significant difference to the fluorescence data. We then ran the plate reader on the optimum gain setting and the reader set the gain to 76. This was too high in our opinion as the weakest dilution had a lower fluorescence than the pure PBS. We then ran the reader at 37°C and 56 to produce our optimal standard curve.</p>
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<p id="pp">The next day in the lab finished preparing competent cells of E.coli DH5α following the provided protocol.Firstly we tested the OD of the cells using our standard settings (see iGEM 2016 file), we were aiming for 0.4-0.5, the reading came out at an avg of 0.2. We waited another 15 minutes as they should have been growing quickly. Taking another OD reading and it had increase to an average of 2.5. It was decided that the reader was giving a strange measurement and we continued with the protocol.The cells were spun down and re-suspended in TF-1 and TF-2 buffer.100µl was aliquoted into 1 ml Eppendorf tubes and these were immediately dropped into liquid nitrogen. The cells were then left in the -80°C freezer. Chloramphenicol was made as a stock of 25mg/ml (weight measured was 76.6mg). This was added to LB media to make our plates for the iGEM interlab parts and so were ready to begin transforming our competent cells with the provided devices.</p>
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<p id="pp">The InterLab measurement kit contained 5 devices; (1) J23101+I13504, (2) J23106+I13504, (3) J23117+I13504, Positive and Negative control. Upon removing these from our freezer and attempting to transform 5µL of each device into our competent cells we found we had no liquid in the tubes provided. They were re-suspended using elution buffer from a Qiagen mini-prep kit. A transformation was performed using heat shock to competent cells, as these were known to be competent. A transformation of plasmids used in the lab that had a strong promoter and GFP was performed in the same way into the competent cells. This was to determine if they were competent. Spread plates were made and incubated at 37°C.</p>
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<p id="pp">We checked our plates the next day to find the competent cells showing strong expression of GFP, all colonies were fluorescing strongly. However the competent cells transformed with the iGEM parts showed no growth and so a new interlab kit was ordered from iGEM.</p>
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<p id="pp">New plasmid constructs arrived and we were instructed to obtain positive and negative cotrols from the kit plate provided. We resuspended DNA from the iGEM registry plates in 10μl of MQ water and then added 5μl constructs (plasmid 1, 2 and 3), 1μl  controls to competent cells, and left one unchanged competent cell as a control, then transformed our competent cells and plated onto 80μl/ml chloramphenicol plates. </p>
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<p id="pp">All colonies were successfully transformed however the positive and negative controls had weak intensity. Positive control had the fewest colonies and fluorescence intensity ranking strongest to weakest for constructs followed the pattern: construct 1, construct 2, construct 3. Consequently 5ml overnights of all successful transformations were produced and left to grow overnight in the 37℃, 220 rpm shaking incubator.</p>
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<p id="pp">The next day involved cell growth, sampling and assaying. The cultures were removed from the incubator and 100μl of each overnight was pipetted into the 96 well plates to read OD at the calibration setting.</p>
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<br> <br>
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<table id="pp" style="width:50%;margin-left:500px;">
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  <tr>
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    <th>Contruct</th>
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    <th>Starting OD</th>
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  </tr>
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    <td>P1</td>
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    <td>0.701</td>
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  </tr>
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  <tr>
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    <td>P2</td>
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    <td>0.614</td>
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  <tr>
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    <td>P3</td>
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    <td>0.615</td>
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  </tr>
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    <td>Positive</td>
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    <td>0.587</td>
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  </tr>
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  <tr>
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    <td>Negative</td>
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    <td>0.593</td>
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  </tr>
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</table>
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<br> <br>
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<p id="pp">The cultures were diluted to achieve an OD600 of 0.02 and incubated at  37℃, 220 rpm .
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Measurements were then taken each hour for 6 hours by pipetting 100µl of each culture into the 96 well plate according to the iGEM layout for Abs600 and Fluorescence measurement and placing this into the calibrated Tecan spectrometer.</p>
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<p id="pp">In addition to this, our team decided to measure the cultures using a latin rectangle arrangement in another 96 well plate. Our lab robot was used to pipette out the corrosponding cultures to their wells according to our preset programme linked to a descrambling spreadsheet. The measurements were taken using the same calibration settings on the Tecan plate reader every hour for 6 hours. The plate was placed into mini vibrating incubator at 37°C each time in between plate readings. A scratch was noticed on the plate lid so we replaced it with a different lid. This lid was cold compared to the incubated plate and so condensation formed this could have affected the results. Placed in incubator for 1.46 mins to warm up.</p>
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Revision as of 15:38, 8 September 2016