Difference between revisions of "Team:UPF CRG Barcelona/InterLab"

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<html>
 
<html>
 
  <link rel="stylesheet" href="https://2018.igem.org/Team:UPF_CRG_Barcelona/styles?action=raw&ctype=text/css" type='text/css'>
 
  <link rel="stylesheet" href="https://2018.igem.org/Team:UPF_CRG_Barcelona/styles?action=raw&ctype=text/css" type='text/css'>
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<script src="https://2018.igem.org/Team:UPF_CRG_Barcelona/plotly?action=raw&ctype=text/javascript" charset="utf-8" type="text/javascript"></script>
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<body>
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<body>
 
   <div class="sticky-top">
 
   <div class="sticky-top">
  
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           <div class="navbar-grid">
 
           <div class="navbar-grid">
 
             <!--class is-active for showing active menu-->
 
             <!--class is-active for showing active menu-->
−
             <a class="navbar-item" href="https://2018.igem.org/Team:UPF_CRG_Barcelona/Description">Project</a>
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             <a style="color: rgb(242, 242, 242)" class="navbar-item" href="https://2018.igem.org/Team:UPF_CRG_Barcelona/Description">Project</a>
−
             <a class="navbar-item" href="https://2018.igem.org/Team:UPF_CRG_Barcelona/DryLab">DryLab</a>
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             <a style="color: rgb(242, 242, 242)" class="navbar-item" href="https://2018.igem.org/Team:UPF_CRG_Barcelona/DryLab">DryLab</a>
−
             <a class="navbar-item" href="https://2018.igem.org/Team:UPF_CRG_Barcelona/Parts">Parts</a>
+
             <a style="color: rgb(242, 242, 242)" class="navbar-item" href="https://2018.igem.org/Team:UPF_CRG_Barcelona/Parts">Parts</a>
 
             <a class="navbar-item is-active" href="https://2018.igem.org/Team:UPF_CRG_Barcelona/Deliverables">Deliverables</a>
 
             <a class="navbar-item is-active" href="https://2018.igem.org/Team:UPF_CRG_Barcelona/Deliverables">Deliverables</a>
−
             <a class="navbar-item" href="https://2018.igem.org/Team:UPF_CRG_Barcelona/WetLab">WetLab</a>
+
             <a style="color: rgb(242, 242, 242)" class="navbar-item" href="https://2018.igem.org/Team:UPF_CRG_Barcelona/WetLab">WetLab</a>
−
             <a class="navbar-item" href="https://2018.igem.org/Team:UPF_CRG_Barcelona/Intro_HP">Human Practices</a>
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             <a style="color: rgb(242, 242, 242)" class="navbar-item" href="https://2018.igem.org/Team:UPF_CRG_Barcelona/Intro_HP">Human Practices</a>
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             <a class="navbar-item" href="https://2018.igem.org/Team:UPF_CRG_Barcelona/Aboutteam">Team</a>
+
             <a style="color: rgb(242, 242, 242)" class="navbar-item" href="https://2018.igem.org/Team:UPF_CRG_Barcelona/Aboutteam">Team</a>
 
           </div>
 
           </div>
 
         </div>
 
         </div>
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   <main>
 
   <main>
  
−
     <section class="container">
+
     <section>
  
 
       <div>
 
       <div>
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         <p class="subapart2">Calibration</p>
 
         <p class="subapart2">Calibration</p>
 
         <p class="subapart3"> OD600 Reference Point</p>
 
         <p class="subapart3"> OD600 Reference Point</p>
−
         <center>
+
         <center style="margin: auto">
−
           <table style="width: 60%;">
+
           <table class="fluid-table" style="max-width: 100%;">
−
             <tr>
+
             <thead>
−
               <th></th>
+
               <tr>
−
              <th>Ludox CL-X</th>
+
                <th> </th>
−
              <th>H<sub>2</sub>O</th>
+
                <th>Ludox CL-X</th>
−
            </tr>
+
                <th>H<sub>2</sub>O</th>
 +
              </tr>
 +
            </thead>
 
             <tr>
 
             <tr>
−
               <td>Replicate 1</td>
+
               <td class="heading" data-label=" "><b>Replicate 1</b></td>
−
               <td>0.056</td>
+
               <td data-label="Ludox CL-X">0.056</td>
−
               <td>0.034</td>
+
               <td data-label="H2O">0.034</td>
 
             </tr>
 
             </tr>
 
             <tr>
 
             <tr>
−
               <td>Replicate 2</td>
+
               <td class="heading" data-label=" "><b>Replicate 2</b></td>
−
               <td>0.056</td>
+
               <td data-label="Ludox CL-X">0.056</td>
−
               <td>0.034</td>
+
               <td data-label="H2O">0.034</td>
 
             </tr>
 
             </tr>
 
             <tr>
 
             <tr>
−
               <td>Replicate 3</td>
+
               <td class="heading" data-label=""><b>Replicate 3</b></td>
−
               <td>0.056</td>
+
               <td data-label="Ludox CL-X">0.056</td>
−
               <td>0.034</td>
+
               <td data-label="H2O">0.034</td>
 
             </tr>
 
             </tr>
 
             <tr>
 
             <tr>
−
               <td>Replicate 4</td>
+
               <td class="heading" data-label=""><b>Replicate 4</b></td>
−
               <td>0.056</td>
+
               <td data-label="Ludox CL-X">0.056</td>
−
               <td>0.034</td>
+
               <td data-label="H2O">0.034</td>
 
             </tr>
 
             </tr>
 
             <tr>
 
             <tr>
−
               <td>Arithmetic Mean</td>
+
               <td class="heading" data-label=""><b>Arithmetic Mean</b></td>
−
               <td>0.056</td>
+
               <td data-label="Ludox CL-X">0.056</td>
−
               <td>0.034</td>
+
               <td data-label="H2O">0.034</td>
 
             </tr>
 
             </tr>
 
             <tr>
 
             <tr>
−
               <td>Corrected Abs600</td>
+
               <td class="heading" data-label=""><b>Corrected Abs600</b></td>
−
               <td>0.022</td>
+
               <td data-label="Ludox CL-X">0.022</td>
−
               <td></td>
+
               <td data-label="H2O">-</td>
 
             </tr>
 
             </tr>
 
             <tr>
 
             <tr>
−
               <td>Reference OD600</td>
+
               <td class="heading" data-label=""><b>Reference OD600</b></td>
−
               <td>0.063</td>
+
               <td data-label="Ludox CL-X">0.063</td>
−
               <td></td>
+
               <td data-label="H2O">-</td>
 
             </tr>
 
             </tr>
 
             <tr>
 
             <tr>
−
               <td>OD600/Abs600</td>
+
               <td class="heading" data-label=""><b>OD600/Abs600</b></td>
−
               <td>2.864</td>
+
               <td data-label="Ludox CL-X">2.864</td>
−
               <td></td>
+
               <td data-label="H2O">-</td>
 
             </tr>
 
             </tr>
 
           </table>
 
           </table>
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         <p class="subapart3">Particle Standard Curve</p>
 
         <p class="subapart3">Particle Standard Curve</p>
 
         <center>
 
         <center>
−
           <div id="interlab_standardcurve_1" style="width:90%;height:400px;">
+
           <div id="interlab_standardcurve_1" style="width:70vw;">
 
             <script>
 
             <script>
 
               INTERLAB = document.getElementById('interlab_standardcurve_1');
 
               INTERLAB = document.getElementById('interlab_standardcurve_1');
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                   family: "PT Sans",
 
                   family: "PT Sans",
 
                   size: 15,
 
                   size: 15,
 +
                  autosize: true // set autosize to rescale
 
                 },
 
                 },
 
                 xaxis: {
 
                 xaxis: {
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           </div>
 
           </div>
 
           <p class="fig-caption">Figure 1 | Graph showing the Standard curve for the calibration of OD600nm with silica beads. </p>
 
           <p class="fig-caption">Figure 1 | Graph showing the Standard curve for the calibration of OD600nm with silica beads. </p>
−
           <div id="interlab_standardcurve" style="width:90%;height:400px;">
+
           <div id="interlab_standardcurve" style="width:70vw;">
 
             <script>
 
             <script>
 
              
 
              
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                 1840000.000,
 
                 1840000.000,
 
                 919000.000, 460000.000, 230000.000, 0.000
 
                 919000.000, 460000.000, 230000.000, 0.000
−
               ],
+
               ];
 
               var y = [0.364, 0.2165, 0.1515, 0.101, 0.073249996, 0.05725, 0.047250003, 0.041, 0.039249998, 0.038,
 
               var y = [0.364, 0.2165, 0.1515, 0.101, 0.073249996, 0.05725, 0.047250003, 0.041, 0.039249998, 0.038,
 
                 0.0375,
 
                 0.0375,
 
                 0.03475
 
                 0.03475
−
               ],
+
               ];
 
               var trace1 = {
 
               var trace1 = {
 
                 x: x,
 
                 x: x,
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                   family: "PT Sans",
 
                   family: "PT Sans",
 
                   size: 15,
 
                   size: 15,
 +
                  autosize: true // set autosize to rescale
 
                 },
 
                 },
 
                 xaxis: {
 
                 xaxis: {
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         </center>
 
         </center>
 
         <center>
 
         <center>
−
           <table style="width: 90%;">
+
           <table class="fluid-table"  style="max-width: 100%;">
−
             <tr>
+
             <thead>
−
               <th>Number of Particles</th>
+
               <tr>
−
              <th>2.35E+08</th>
+
                <th>Number of Particles</th>
−
              <th>1.18E+08</th>
+
                <th>2.35e8</th>
−
              <th>5.88E+07</th>
+
                <th>1.18e8</th>
−
              <th>2.94E+07</th>
+
                <th>5.88e7</th>
−
              <th>1.47E+07</th>
+
                <th>2.94e7</th>
−
              <th>7.35E+06</th>
+
                <th>1.47e7</th>
−
              <th>3.68E+06</th>
+
                <th>7.35e6</th>
−
              <th>1.84E+06</th>
+
                <th>3.68e6</th>
−
              <th>9.19E+05</th>
+
                <th>1.84e6</th>
−
              <th>4.60E+05</th>
+
                <th>9.19e5</th>
−
              <th>2.30E+05</th>
+
                <th>4.60e5</th>
−
            </tr>
+
                <th>2.30e5</th>
 +
              </tr>
 +
            </thead>
 
             <tr>
 
             <tr>
−
               <td>Mean particles / Abs600</td>
+
               <td class="heading" data-label="Number of Particles"><b>Mean particles / Abs600</b></td>
−
               <td>7.15E+08</td>
+
               <td data-label="2.35E+08">7.15e8</td>
−
               <td>6.47E+08</td>
+
               <td data-label="1.18E+08">6.47e8</td>
−
               <td>5.04E+08</td>
+
               <td data-label="5.88E+07">5.04e8</td>
−
               <td>4.44E+08</td>
+
               <td data-label="2.94E+07">4.44e8</td>
−
               <td>3.82E+08</td>
+
               <td data-label="1.47E+07">3.82e8</td>
−
               <td>3.27E+08</td>
+
               <td data-label="7.35E+06">3.27e8</td>
−
               <td>2.94E+08</td>
+
               <td data-label="3.68E+06">2.94e8</td>
−
               <td>2.94E+08</td>
+
               <td data-label="1.84E+06">2.94e8</td>
−
               <td>2.04E+08</td>
+
               <td data-label="9.19E+05">2.04e8</td>
−
               <td>1.41E+08</td>
+
               <td data-label="4.60E+05">1.41e8</td>
−
               <td>8.36E+07</td>
+
               <td data-label="2.30E+05">8.36e7</td>
 
             </tr>
 
             </tr>
 
             <tr>
 
             <tr>
−
               <td>Mean of med-high levels:</td>
+
               <td class="heading" data-label="Number of Particles"><b>Mean of med-high levels</b></td>
−
               <td></td>
+
               <td data-label="2.35E+08">-</td>
−
               <td>4.61E+08</td>
+
               <td data-label="1.18E+08">4.61e8</td>
−
               <td></td>
+
               <td data-label="5.88E+07">-</td>
−
               <td></td>
+
               <td data-label="2.94E+07">-</td>
−
               <td></td>
+
               <td data-label="1.47E+07">-</td>
−
               <td></td>
+
               <td data-label="7.35E+06">-</td>
−
               <td></td>
+
               <td data-label="3.68E+06">-</td>
−
               <td></td>
+
               <td data-label="1.84E+06">-</td>
−
               <td></td>
+
               <td data-label="9.19E+05">-</td>
−
               <td></td>
+
               <td data-label="4.60E+05">-</td>
−
               <td></td>
+
               <td data-label="2.30E+05">-</td>
 
             </tr>
 
             </tr>
 
           </table>
 
           </table>
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         <p>This calibration has a R<sup>2</sup>=0,8547.</p>
 
         <p>This calibration has a R<sup>2</sup>=0,8547.</p>
 
         <center>
 
         <center>
−
           <div id="interlab_standardcurve3" style="width:90%;height:400px;">
+
           <div id="interlab_standardcurve3" style="width:70vw;">
 
             <script>
 
             <script>
 
               var x = [10, 5, 2.5, 0.625, 0.3125, 0.15625, 0.078125, 0.0390625, 0.0195313, 0.0097656, 0];
 
               var x = [10, 5, 2.5, 0.625, 0.3125, 0.15625, 0.078125, 0.0390625, 0.0195313, 0.0097656, 0];
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                   family: "PT Sans",
 
                   family: "PT Sans",
 
                   size: 15,
 
                   size: 15,
 +
                  autosize: true // set autosize to rescale
 
                 },
 
                 },
 
                 xaxis: {
 
                 xaxis: {
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           <p class="fig-caption">Figure 3 | Fluorescence standard curve to calibrate the measurements with fluorescein. </p>
 
           <p class="fig-caption">Figure 3 | Fluorescence standard curve to calibrate the measurements with fluorescein. </p>
 
          
 
          
−
           <div id="interlab_standardcurve_4" style="width:90%;height:400px;">
+
           <div id="interlab_standardcurve_4" style="width:70vw;">
 
             <script>
 
             <script>
 
              
 
              
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                   family: "PT Sans",
 
                   family: "PT Sans",
 
                   size: 15,
 
                   size: 15,
 +
                  autosize: true // set autosize to rescale
 
                 },
 
                 },
 
                 xaxis: {
 
                 xaxis: {
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         </center>
 
         </center>
 
         <center>
 
         <center>
−
           <table style="width: 90%;">
+
           <table class="fluid-table"  style="max-width: 90%;">
 +
            <thead>
 +
              <tr>
 +
                <th>Fluorescein uM</th>
 +
                <th>10.00</th>
 +
                <th>5.00</th>
 +
                <th>2.50</th>
 +
                <th>1.25</th>
 +
                <th>0.625</th>
 +
                <th>0.3125</th>
 +
                <th>0.15625</th>
 +
                <th>0.078125</th>
 +
                <th>0.0390625</th>
 +
                <th>0.0295313</th>
 +
                <th>0.0097656</th>
 +
              </tr>
 +
            </thead>
 
             <tr>
 
             <tr>
−
               <th>Fluorescein uM</th>
+
               <td class="heading" data-label="Fluorescein uM"><b>uM Fluorescein / a.u.</b></td>
−
               <th>10.00</th>
+
               <td data-label="10.00">1.31E-04</td>
−
               <th>5.00</th>
+
               <td data-label="5.00">6,57E-05</td>
−
               <th>2.50</th>
+
               <td data-label="2.50">6.18E-05</td>
−
               <th>1.25</th>
+
               <td data-label="1.25">6.00E-05</td>
−
               <th>0.625</th>
+
               <td data-label="0.625">5.91E-05</td>
−
               <th>0.3125</th>
+
               <td data-label="0.3125">5.84E-05</td>
−
               <th>0.15625</th>
+
               <td data-label="0.15625">5.81E-05</td>
−
               <th>0.078125</th>
+
               <td data-label="0.078125">7.17E-05</td>
−
               <th>0.0390625</th>
+
               <td data-label="0.0390625">5.01E-05</td>
−
               <th>0.0295313</th>
+
               <td data-label="0.0295313">4.81E-05</td>
−
               <th>0.0097656</th>
+
               <td data-label="0.0097656">4.85E-05</td>
 
             </tr>
 
             </tr>
 
             <tr>
 
             <tr>
−
               <td>uM Fluorescein / a.u.</td>
+
               <td class="heading" data-label="Fluorescein uM"><b>Mean uM fluorescein / a.u.</b></td>
−
               <td>1.31E-04</td>
+
               <td data-label="10.00">-</td>
−
               <td>6,57E-05</td>
+
               <td data-label="5.00">6.10E-05</td>
−
               <td>6.18E-05</td>
+
               <td data-label="2.50">-</td>
−
               <td>6.00E-05</td>
+
               <td data-label="1.25">-</td>
−
               <td>5.91E-05</td>
+
               <td data-label="0.625">-</td>
−
               <td>5.84E-05</td>
+
               <td data-label="0.3125">-</td>
−
               <td>5.81E-05</td>
+
               <td data-label="0.15625">-</td>
−
               <td>7.17E-05</td>
+
               <td data-label="0.078125">-</td>
−
               <td>5.01E-05</td>
+
               <td data-label="0.0390625">-</td>
−
               <td>4.81E-05</td>
+
               <td data-label="0.0295313">-</td>
−
               <td>4.85E-05</td>
+
               <td data-label="0.0097656">-</td>
 
             </tr>
 
             </tr>
 
             <tr>
 
             <tr>
−
               <td>Mean uM fluorescein / a.u.</td>
+
               <td class="heading" data-label="Fluorescein uM"><b>MEFL / a.u.</b></td>
−
              <td></td>
+
               <td data-label="10.00">-</td>
−
               <td>6.10E-05</td>
+
               <td data-label="5.00">3.67E+08</td>
−
               <td></td>
+
               <td data-label="2.50">-</td>
−
              <td></td>
+
               <td data-label="1.25">-</td>
−
              <td></td>
+
               <td data-label="0.625">-</td>
−
              <td></td>
+
               <td data-label="0.3125">-</td>
−
              <td></td>
+
               <td data-label="0.15625">-</td>
−
              <td></td>
+
               <td data-label="0.078125">-</td>
−
              <td></td>
+
               <td data-label="0.0390625">-</td>
−
              <td></td>
+
               <td data-label="0.0295313">-</td>
−
              <td></td>
+
               <td data-label="0.0097656">-</td>
−
            </tr>
+
−
            <tr>
+
−
              <td>MEFL / a.u.</td>
+
−
              <td></td>
+
−
              <td>3.67E+08</td>
+
−
               <td></td>
+
−
               <td></td>
+
−
               <td></td>
+
−
               <td></td>
+
−
               <td></td>
+
−
               <td></td>
+
−
               <td></td>
+
−
               <td></td>
+
−
               <td></td>
+
 
             </tr>
 
             </tr>
 
           </table>
 
           </table>
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         <p class="subapart3">Fluorescence per optical density</p>
 
         <p class="subapart3">Fluorescence per optical density</p>
 
         <center>
 
         <center>
−
           <div id="interlab_measure1" style="width:90%;height:400px;"></div>
+
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           <script>
 
           <script>
 
             var x = ['Negative Control', 'Negative Control', 'Negative Control', 'Negative Control', 'Negative Control',
 
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           <p class="fig-caption">Figure 5 | Fluorescein (uM) related to OD600nm to extrapolate measures of fluorescence with optical density measurements.  </p>
 
           <p class="fig-caption">Figure 5 | Fluorescein (uM) related to OD600nm to extrapolate measures of fluorescence with optical density measurements.  </p>
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           <div id="interlab_measure2" style="width:70vw;">
 
             <script>
 
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               var x = ['Negative Control', 'Negative Control', 'Negative Control', 'Negative Control',
 
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         <p class="subapart3">Colony Forming Units per OD600</p>
 
         <p class="subapart3">Colony Forming Units per OD600</p>
 
         <center>
 
         <center>
−
           <div id="interlab_measure3" style="width:90%;height:400px;">
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Latest revision as of 00:32, 18 October 2018

Wiki

INTERLAB

Introduction

The aim of the Interlab Study is to develop a reliable and repeatable measurement based on cell number, fluorescence, absorbance (optical density) and colony formation units (CFUs). Each year, iGEM teams collaborate in measuring these parameters following the same protocol to obtain a way to have accurate and reliable measurements, which are essential for all sciences, including synthetic biology.

The main part of the Interlab has been always the green fluorescent protein (GFP), one of the biological markers most used in synthetic biology.

The goal of the fifth edition of Interlab is to discover the sources of variability in measurements and be able to correct them, so the measurements taken in different labs will not be variable anymore.

This year question is: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or CFUs instead of OD?

Materials and Methods

Before the starting the experimental part, a plate reader was needed. Due to the lack of plate reader in our laboratory, it was kindly asked to Proteomics and Protein Chemistry Unit (DCEXS-UPF, PRBB) to use their equipment. The plate reader is the Synergy HTX Multimode Reader from Biotek, it can measure both absorbance and fluorescence. It has pathlength correction, which was not disconnected. It has control over temperature and it was set as room temperature (around 24-25ºC). The excitation filter was 485/20 nm and the emission filter 528/20 nm and bottom optics were used. Moreover, the plates were black and flat-bottomed.

Eight plasmids needed to be characterized in DH5-alpha E.coli strain in order to follow the protocol. The strain was obtained by collaboration with BIO-IQS iGEM team. The plasmids are the following: BBa_R0040, BBa_R0040, BBa_I20270, BBa_J3604000, BBA_J364001, BBa_J364002, BBa_J364007, BBa_J364008, BBa_J364009.

The materials used over the protocol are the same ones specified in the iGEM 2018 Interlab Study Protocol.

Results

Calibration

OD600 Reference Point

Ludox CL-X H2O
Replicate 1 0.056 0.034
Replicate 2 0.056 0.034
Replicate 3 0.056 0.034
Replicate 4 0.056 0.034
Arithmetic Mean 0.056 0.034
Corrected Abs600 0.022 -
Reference OD600 0.063 -
OD600/Abs600 2.864 -

Table 1 | Date from the OD600 Reference Point in calibration 1.

Particle Standard Curve

Figure 1 | Graph showing the Standard curve for the calibration of OD600nm with silica beads.

Figure 2 | Graph showing the Logaritmic Standard curve for the calibration of OD600nm with silica beads.

Number of Particles 2.35e8 1.18e8 5.88e7 2.94e7 1.47e7 7.35e6 3.68e6 1.84e6 9.19e5 4.60e5 2.30e5
Mean particles / Abs600 7.15e8 6.47e8 5.04e8 4.44e8 3.82e8 3.27e8 2.94e8 2.94e8 2.04e8 1.41e8 8.36e7
Mean of med-high levels - 4.61e8 - - - - - - - - -

Table 2 | Particles per Optical density measured.

Fluorescein Standard Curve

This calibration has a R2=0,8547.

Figure 3 | Fluorescence standard curve to calibrate the measurements with fluorescein.

Figure 4 | Logaritmic Fluorescence Standard curve to calibrate the measurements with fluorescein.

Fluorescein uM 10.00 5.00 2.50 1.25 0.625 0.3125 0.15625 0.078125 0.0390625 0.0295313 0.0097656
uM Fluorescein / a.u. 1.31E-04 6,57E-05 6.18E-05 6.00E-05 5.91E-05 5.84E-05 5.81E-05 7.17E-05 5.01E-05 4.81E-05 4.85E-05
Mean uM fluorescein / a.u. - 6.10E-05 - - - - - - - - -
MEFL / a.u. - 3.67E+08 - - - - - - - - -

Table 3 | Fluorescence in arbitrary units (a.u.) represented by Molecules of Equivalent FLuorescein (MEFL).

Measurement

Fluorescence per optical density

Figure 5 | Fluorescein (uM) related to OD600nm to extrapolate measures of fluorescence with optical density measurements.

Figure 6 | Fluorescence per particle measured at 0 hours and at 6 hours after growing at 37ºC

Colony Forming Units per OD600

Figure 7 | Colony forming units count on each dilution and on each device (postive and negative control)