Difference between revisions of "Team:Harvard/InterLab"

 
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<h1>InterLab</h1>
 
<h1>InterLab</h1>
  
 
<h3>Overview</h3>
 
<h3>Overview</h3>
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<p>
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 +
<br>
 
Difficulty in taking reliable and reproducible measurements remains a key obstacle in establishing synthetic biology as an engineering discipline. The InterLab Study is part of the Measurement Committee’s continuing effort to develop a standard measurement procedure for green fluorescent protein (GFP). Despite being one of the most commonly used markers in synthetic biology, labs often resort to making relative comparisons, which makes it difficult for labs to share and data and/or constructs.  
 
Difficulty in taking reliable and reproducible measurements remains a key obstacle in establishing synthetic biology as an engineering discipline. The InterLab Study is part of the Measurement Committee’s continuing effort to develop a standard measurement procedure for green fluorescent protein (GFP). Despite being one of the most commonly used markers in synthetic biology, labs often resort to making relative comparisons, which makes it difficult for labs to share and data and/or constructs.  
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<br><br>
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<p>
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For the fourth installment of the InterLab, iGEM hopes to establish a GFP measurement protocol that can be used to produce comparable GFP measurements on different plate readers. In addition, iGEM teams from around the world have tested RBS devices (BCDs) intended to increase the precision and reliability of gene expression. The parts used include six test devices  
 
For the fourth installment of the InterLab, iGEM hopes to establish a GFP measurement protocol that can be used to produce comparable GFP measurements on different plate readers. In addition, iGEM teams from around the world have tested RBS devices (BCDs) intended to increase the precision and reliability of gene expression. The parts used include six test devices  
 
(<a href="http://parts.igem.org/Part:BBa_J364000">BBa_J364000</a>,  
 
(<a href="http://parts.igem.org/Part:BBa_J364000">BBa_J364000</a>,  
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<a href="http://parts.igem.org/Part:BBa_J364004">BBa_J364004</a>,  
 
<a href="http://parts.igem.org/Part:BBa_J364004">BBa_J364004</a>,  
 
<a href="http://parts.igem.org/Part:BBa_J364005">BBa_J364005</a>), as well as a positive (<a href="http://parts.igem.org/Part:BBa_I20270">BBa_I20270</a>) and negative (<a href="http://parts.igem.org/Part:BBa_R0040">BBa_R0040</a>) control. All parts are located in the pSB1C3 plasmid and carry chloramphenicol resistance.  
 
<a href="http://parts.igem.org/Part:BBa_J364005">BBa_J364005</a>), as well as a positive (<a href="http://parts.igem.org/Part:BBa_I20270">BBa_I20270</a>) and negative (<a href="http://parts.igem.org/Part:BBa_R0040">BBa_R0040</a>) control. All parts are located in the pSB1C3 plasmid and carry chloramphenicol resistance.  
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</p>
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<br>
  
 
<h3>Methods and Materials</h3>
 
<h3>Methods and Materials</h3>
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     <div id="collapse1" class="panel-collapse collapse">
 
     <div id="collapse1" class="panel-collapse collapse">
 
       <div class="panel-body">
 
       <div class="panel-body">
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         <h5>Materials</h5>
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        <p>Plate Reader: Synergy H1</p>
 +
         <h5>Abs OD600</h5>
 
         <ul>
 
         <ul>
−
           <li>1 mL LUDOX</li>
+
           <li>Wavelengths: 600</li>
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           <li>H2O</li>
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           <li>Read Speed: Normal</li>
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           <li>96 well plate</li>
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           <li>Delay: 100 msec</li>
 +
        </ul>
 +
        <h5>Fluorescence</h5>
 +
        <ul>
 +
          <li>Excitation: 395</li>
 +
          <li>Emission: 509</li>
 +
          <li>Optics: Top</li>
 +
          <li>Gain: 75</li>
 +
          <li>Light Source: Xenon Flash</li>
 +
          <li>Lamp Energy: High</li>
 +
          <li>Read Speed: Normal</li>
 +
          <li>Delay: 100 msec</li>
 +
          <li>Read Height: 7 mm</li>
 
         </ul>
 
         </ul>
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        <h5>Methods</h5>
 
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        <ol>
 
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          <li>100 uL of LUDOX-S40 from the InterLab Measurement Kit added into wells A1, B1, C1, and D1</li>
 
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          <li>100 uL of dH2O added to wells A2, B2, C2, D2</li>
 
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          <li>Absorbance at 600 nm taken for all samples in setup described above</li>
 
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          <li>Data used to obtain correction factor</li>
 
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        </ol>
 
 
       </div>
 
       </div>
 
     </div>
 
     </div>
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         <h5>Materials</h5>
 
         <h5>Materials</h5>
 
         <ul>
 
         <ul>
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           <li>1 mL LUDOX</li>
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           <li>Fluorescein</li>
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           <li>H2O</li>
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           <li>10 mL 1xPBS (phosphate buffered saline)</li>
 
           <li>96 well plate</li>
 
           <li>96 well plate</li>
 
         </ul>
 
         </ul>
 
         <h5>Methods</h5>
 
         <h5>Methods</h5>
 
         <ol>
 
         <ol>
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           <li>100 uL of LUDOX-S40 from the InterLab Measurement Kit added into wells A1, B1, C1, and D1</li>
+
           <li>Fluorescein stock tube from InterLab Measurement Kit spun down to make sure pellet is at bottom of tube</li>
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          <li>100 uL of dH2O added to wells A2, B2, C2, D2</li>
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          <li>2x fluorescein stock solution (100 uM) prepared by resuspending fluorescein in 1 mL of 1xPBS</li>
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           <li>Absorbance at 600 nm taken for all samples in setup described above</li>
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          <li>500 uL of 2x fluorescein stock solution diluted with 500 uL of 1xPBS to make 1mL of 50 uM (1x) fluorescein solution</li>
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           <li>Data used to obtain correction factor</li>
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          <li>Serial dilutions of fluorescein: <br>
 +
              <ol type="a">
 +
                <li>100 uL of PBS added into wells A2, B2, C2, D2... A12, B12</li>
 +
                <li>200 uL of fluorescein 1x stock solution added to A1, B1, C1, D1</li>
 +
                <li>100 uL fluorescein stock solution transferred from A1 into A2</li>
 +
                <li>A2 mixed by pipetting up and down, then 100 uL transferred to A3</li>
 +
                <li>A3 mixed by pipetting up and down, then 100 uL transferred to A4</li>
 +
                <li>A4 mixed by pipetting up and down, then 100 uL transferred to A5</li>
 +
                <li>A5 mixed by pipetting up and down, then 100 uL transferred to A6</li>
 +
                <li>A6 mixed by pipetting up and down, then 100 uL transferred to A7</li>
 +
                <li>A8 mixed by pipetting up and down, then 100 uL transferred to A9</li>
 +
                <li>A9 mixed by pipetting up and down, then 100 uL transferred to A10</li>
 +
                <li>A10 mixed by pipetting up and down, then 100 uL transferred to A11</li>
 +
                <li>A11 mixed by pipetting up and down, then 100 uL transferred to liquid waste</li>
 +
              </ol>
 +
           </li>
 +
          <li>Step 4 repeated for rows B-D</li>
 +
          <li>Fluorescence for all samples measured using setup described above</li>
 +
        </ol>
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      </div>
 +
    </div>
 +
  </div>
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 +
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      <h4 class="panel-title">
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        <a data-toggle="collapse" data-parent="#accordion" href="#collapse4">Cell Measurements</a>
 +
      </h4>
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    </div>
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    <div id="collapse4" class="panel-collapse collapse">
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      <div class="panel-body">
 +
        <h5>Materials</h5>
 +
        <ul>
 +
           <li>Competent Cells (<i>Escherichia coli</i> strain DH5 alpha</li>
 +
          <li>LB media</li>
 +
          <li>Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH, working stock 25 ug/mL)</li>
 +
          <li>50 mL Falcon tubes covered in foil</li>
 +
          <li>Incubator at 37ºC</li>
 +
          <li>Devices from InterLab Measurement Kit
 +
            <ul>
 +
              <li>Positive Control</li>
 +
              <li>Negative Control</li>
 +
              <li>Test Device 1: J23101 + I13504</li>
 +
              <li>Test Device 2: J23106 + I13504</li>
 +
              <li>Test Device 3: J23117 + I13504</li>
 +
              <li>Test Device 4: J23101.BCD2.E0040.B0015</li>
 +
              <li>Test Device 5: J23106.BCD2.E0040.B0015</li>
 +
              <li>Test Device 6: J23117.BCD2.E0040.B0015</li>
 +
            </ul>
 +
          </li>
 +
        </ul>
 +
        <h5>Methods</h5>
 +
        <ol>
 +
          <li>Each of the plasmids listed above were located on Plate 7 of the distribution kit and resuspended in 10 uL of dH2O</li>
 +
          <li>Each device was transformed into 10 uL of competent cells (see here for the full transformation protocol)</li>
 +
          <li>Transformations were spot plated onto an agar plate with chloramphenicol and grown overnight(see here for the full spot plating protocol)</li>
 +
          <li>On Day 2, 2 colonies were picked from each plate and inoculated in 10 mL of LB medium + Chloramphenicol in 50 mL Falcon tubes covered with foil. These cultures were grown overnight in an incubator at 37ºC and 220 rpm.</li>
 +
          <li>On Day 3, 100 uL of each culture was pipetted into a 96 well plate for an OD600 reading, which was then used for the Dilution Calculations in the Excel sheet provided by iGEM
 +
          </li>
 +
          <li>Each culture was diluted to a target OD of 0.02 following the preloading culture and media volumes calculated by the Dilution excel sheet (see OD Readings and Dilutions Calculations in the Results section below) in 12 mL of LB media + chloramphenicol in 50 mL falcon tubes wrapped in foil</li>
 +
          <li>Cultures were placed in incubator at 37ºC and 220 rpm</li>
 +
          <li>4 replicates of 100 uL samples were taken from each culture at 0, 2, 4, and 6 hours of incubation and placed in a 96 well plate for OD and fluorescence measurements using the setup described above</li>
 
         </ol>
 
         </ol>
 
       </div>
 
       </div>
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<h3>Results</h3>
 
<h3>Results</h3>
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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2017.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2017.igem.org/Judging/Awards"> award listed above</a>. </p>
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      <h4 class="panel-title">
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2017.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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        <a data-toggle="collapse" data-parent="#accordion" href="#results1">OD600 Calibration</a>
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    <div id="results1" class="panel-collapse collapse">
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        <img src="https://static.igem.org/mediawiki/2017/6/63/Harvard--OD_calibration2.png" class="centerImage" alt="OD Calibration">
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<h1>InterLab</h1>
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    <div class="panel-heading">
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<h3>Bronze Medal Criterion #4</h3>
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      <h4 class="panel-title">
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<p><b>Standard Tracks:</b> Participate in the Interlab Measurement Study and/or improve the characterization of an existing BioBrick Part or Device and enter this information on that part's Main Page in the Registry. The part that you are characterizing must NOT be from a 2017 part number range.
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        <a data-toggle="collapse" data-parent="#accordion" href="#results2">Fluorescein Standard Curve</a>
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<br><br>
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      </h4>
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For teams participating in the <a href="https://2017.igem.org/Competition/InterLab_Study">InterLab study</a>, all work must be shown on this page.
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    <div id="results2" class="panel-collapse collapse">
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        <img src="https://static.igem.org/mediawiki/2017/2/27/Harvard--fluorescein_standard2.png" class="centerImage" alt="Fluorescein Standard Curve">
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        <a data-toggle="collapse" data-parent="#accordion" href="#results3">Dilution Calculations</a>
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      </h4>
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    </div>
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    <div id="results3" class="panel-collapse collapse">
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      <div class="panel-body">
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<table class="table table-bordered table-hover table-responsive">
 +
              <thead>
 +
                <tr>
 +
                  <th>Sample</th>
 +
                  <th>Abs600 Reading</th>
 +
                  <th>Volume of Preloading Culture (mL)</th>
 +
                  <th>Volume of Preloading Media (mL)</th>
 +
                </tr>
 +
              </thead>
 +
              <tbody>
 +
                <tr>
 +
                  <td>Positive control (1)</td>
 +
                  <td>1.011</td>
 +
                  <td>0.205</td>
 +
                  <td>9.795</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>Positive control (2)</td>
 +
                  <td>2.261</td>
 +
                  <td>0.0900</td>
 +
                  <td>9.910</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>Negative control</td>
 +
                  <td>1.3</td>
 +
                  <td>0.158</td>
 +
                  <td>9.841</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>Negative control (2)</td>
 +
                  <td>0.734</td>
 +
                  <td>0.287</td>
 +
                  <td>9.713</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>Device 1 (1)</td>
 +
                  <td>0.443</td>
 +
                  <td>1.493</td>
 +
                  <td>8.507</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>Device 1 (2)</td>
 +
                  <td>0.138</td>
 +
                  <td>1.980</td>
 +
                  <td>8.020</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>Device 2 (1)</td>
 +
                  <td>0.425</td>
 +
                  <td>2.515</td>
 +
                  <td>7.485</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>Device 2 (2)</td>
 +
                  <td>0.136</td>
 +
                  <td>2.020</td>
 +
                  <td>7.980</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>Device 3 (1)</td>
 +
                  <td>0.507</td>
 +
                  <td>0.426</td>
 +
                  <td>9.574</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>Device 3 (2)</td>
 +
                  <td>0.543</td>
 +
                  <td>0.395</td>
 +
                  <td>9.604</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>Device 4 (1)</td>
 +
                  <td>0.306</td>
 +
                  <td>0.743</td>
 +
                  <td>9.257</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>Device 4 (2)</td>
 +
                  <td>0.409</td>
 +
                  <td>0.538</td>
 +
                  <td>9.462</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>Device 5 (1)</td>
 +
                  <td>0.195</td>
 +
                  <td>0.266</td>
 +
                  <td>9.734</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>Device 5 (2)</td>
 +
                  <td>0.494</td>
 +
                  <td>0.438</td>
 +
                  <td>9.562</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>Device 6 (1)</td>
 +
                  <td>1.597</td>
 +
                  <td>0.357</td>
 +
                  <td>9.643</td>
 +
                </tr>
 +
                  <td>Device 6 (2)</td>
 +
                  <td>1.58</td>
 +
                  <td>0.130</td>
 +
                  <td>9.870</td>
 +
                </tr>
 +
                <tr>
 +
                  <td>Media+chl</td>
 +
                  <td>0.037</td>
 +
                  <td></td>
 +
                  <td></td>
 +
                </tr>
 +
              </tbody>
 +
            </table>
 +
      </div>
 +
    </div>
 +
  </div>
  
 +
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    <div class="panel-heading">
 +
      <h4 class="panel-title">
 +
        <a data-toggle="collapse" data-parent="#accordion" href="#results4">OD Readings</a>
 +
      </h4>
 +
    </div>
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    <div id="results4" class="panel-collapse collapse">
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      <div class="panel-body">
 +
        <img src="https://static.igem.org/mediawiki/2017/2/2f/Harvard--OD_readings3.png" class="centerImage" alt="OD Readings">
 +
        <p style="text-align:center">Averaged OD measurements of the 4 replicates for each sample, taken at two hour intervals. </p>
 +
      </div>
 +
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 +
      <h4 class="panel-title">
 +
        <a data-toggle="collapse" data-parent="#accordion" href="#results5">Fluorescence Readings</a>
 +
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 +
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 +
    <div id="results5" class="panel-collapse collapse">
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      <div class="panel-body">
 +
        <img src="https://static.igem.org/mediawiki/2017/f/f6/Harvard--fluor_readings.png" class="centerImage" alt="Fluorescence Readings">
 +
        <p style="text-align:center">Averaged fluorescence measurements of the 4 replicates for each sample, taken at two hour intervals.</p>
 +
      </div>
 +
    </div>
 +
  </div>
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Latest revision as of 17:54, 31 October 2017

Harvard OPTI POLY

InterLab

Overview


Difficulty in taking reliable and reproducible measurements remains a key obstacle in establishing synthetic biology as an engineering discipline. The InterLab Study is part of the Measurement Committee’s continuing effort to develop a standard measurement procedure for green fluorescent protein (GFP). Despite being one of the most commonly used markers in synthetic biology, labs often resort to making relative comparisons, which makes it difficult for labs to share and data and/or constructs.

For the fourth installment of the InterLab, iGEM hopes to establish a GFP measurement protocol that can be used to produce comparable GFP measurements on different plate readers. In addition, iGEM teams from around the world have tested RBS devices (BCDs) intended to increase the precision and reliability of gene expression. The parts used include six test devices (BBa_J364000, BBa_J364001, BBa_J364002, BBa_J364003, BBa_J364004, BBa_J364005), as well as a positive (BBa_I20270) and negative (BBa_R0040) control. All parts are located in the pSB1C3 plasmid and carry chloramphenicol resistance.

Methods and Materials

Plate Reader Setup

Plate Reader: Synergy H1

Abs OD600
  • Wavelengths: 600
  • Read Speed: Normal
  • Delay: 100 msec
Fluorescence
  • Excitation: 395
  • Emission: 509
  • Optics: Top
  • Gain: 75
  • Light Source: Xenon Flash
  • Lamp Energy: High
  • Read Speed: Normal
  • Delay: 100 msec
  • Read Height: 7 mm

OD600 Reference Point

Materials
  • 1 mL LUDOX
  • H2O
  • 96 well plate
Methods
  1. 100 uL of LUDOX-S40 from the InterLab Measurement Kit added into wells A1, B1, C1, and D1
  2. 100 uL of dH2O added to wells A2, B2, C2, D2
  3. Absorbance at 600 nm taken for all samples in setup described above
  4. Data used to obtain correction factor

Protocol Fluorescein Fluorescence Standard Curve

Materials
  • Fluorescein
  • 10 mL 1xPBS (phosphate buffered saline)
  • 96 well plate
Methods
  1. Fluorescein stock tube from InterLab Measurement Kit spun down to make sure pellet is at bottom of tube
  2. 2x fluorescein stock solution (100 uM) prepared by resuspending fluorescein in 1 mL of 1xPBS
  3. 500 uL of 2x fluorescein stock solution diluted with 500 uL of 1xPBS to make 1mL of 50 uM (1x) fluorescein solution
  4. Serial dilutions of fluorescein:
    1. 100 uL of PBS added into wells A2, B2, C2, D2... A12, B12
    2. 200 uL of fluorescein 1x stock solution added to A1, B1, C1, D1
    3. 100 uL fluorescein stock solution transferred from A1 into A2
    4. A2 mixed by pipetting up and down, then 100 uL transferred to A3
    5. A3 mixed by pipetting up and down, then 100 uL transferred to A4
    6. A4 mixed by pipetting up and down, then 100 uL transferred to A5
    7. A5 mixed by pipetting up and down, then 100 uL transferred to A6
    8. A6 mixed by pipetting up and down, then 100 uL transferred to A7
    9. A8 mixed by pipetting up and down, then 100 uL transferred to A9
    10. A9 mixed by pipetting up and down, then 100 uL transferred to A10
    11. A10 mixed by pipetting up and down, then 100 uL transferred to A11
    12. A11 mixed by pipetting up and down, then 100 uL transferred to liquid waste
  5. Step 4 repeated for rows B-D
  6. Fluorescence for all samples measured using setup described above

Cell Measurements

Materials
  • Competent Cells (Escherichia coli strain DH5 alpha
  • LB media
  • Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH, working stock 25 ug/mL)
  • 50 mL Falcon tubes covered in foil
  • Incubator at 37ºC
  • Devices from InterLab Measurement Kit
    • 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
Methods
  1. Each of the plasmids listed above were located on Plate 7 of the distribution kit and resuspended in 10 uL of dH2O
  2. Each device was transformed into 10 uL of competent cells (see here for the full transformation protocol)
  3. Transformations were spot plated onto an agar plate with chloramphenicol and grown overnight(see here for the full spot plating protocol)
  4. On Day 2, 2 colonies were picked from each plate and inoculated in 10 mL of LB medium + Chloramphenicol in 50 mL Falcon tubes covered with foil. These cultures were grown overnight in an incubator at 37ºC and 220 rpm.
  5. On Day 3, 100 uL of each culture was pipetted into a 96 well plate for an OD600 reading, which was then used for the Dilution Calculations in the Excel sheet provided by iGEM
  6. Each culture was diluted to a target OD of 0.02 following the preloading culture and media volumes calculated by the Dilution excel sheet (see OD Readings and Dilutions Calculations in the Results section below) in 12 mL of LB media + chloramphenicol in 50 mL falcon tubes wrapped in foil
  7. Cultures were placed in incubator at 37ºC and 220 rpm
  8. 4 replicates of 100 uL samples were taken from each culture at 0, 2, 4, and 6 hours of incubation and placed in a 96 well plate for OD and fluorescence measurements using the setup described above

Results

OD600 Calibration

OD Calibration

Fluorescein Standard Curve

Fluorescein Standard Curve

Dilution Calculations

Sample Abs600 Reading Volume of Preloading Culture (mL) Volume of Preloading Media (mL)
Positive control (1) 1.011 0.205 9.795
Positive control (2) 2.261 0.0900 9.910
Negative control 1.3 0.158 9.841
Negative control (2) 0.734 0.287 9.713
Device 1 (1) 0.443 1.493 8.507
Device 1 (2) 0.138 1.980 8.020
Device 2 (1) 0.425 2.515 7.485
Device 2 (2) 0.136 2.020 7.980
Device 3 (1) 0.507 0.426 9.574
Device 3 (2) 0.543 0.395 9.604
Device 4 (1) 0.306 0.743 9.257
Device 4 (2) 0.409 0.538 9.462
Device 5 (1) 0.195 0.266 9.734
Device 5 (2) 0.494 0.438 9.562
Device 6 (1) 1.597 0.357 9.643
Device 6 (2) 1.58 0.130 9.870
Media+chl 0.037

OD Readings

OD Readings

Averaged OD measurements of the 4 replicates for each sample, taken at two hour intervals.

Fluorescence Readings

Fluorescence Readings

Averaged fluorescence measurements of the 4 replicates for each sample, taken at two hour intervals.