Difference between revisions of "Team:Edinburgh/InterLab"

 
(18 intermediate revisions by the same user not shown)
Line 1: Line 1:
{{Edinburgh_practices}}
+
{{Edinburgh_InterLab}}
 
<html>
 
<html>
  
Line 7: Line 7:
 
<script src="https://maxcdn.bootstrapcdn.com/bootstrap/3.3.5/js/bootstrap.min.js"></script>
 
<script src="https://maxcdn.bootstrapcdn.com/bootstrap/3.3.5/js/bootstrap.min.js"></script>
 
     </head>
 
     </head>
 
<body>
 
 
 
  <!-- menu -->  
 
  <!-- menu -->  
      <div class="navbar-wrapper">
+
<div id="custom-bootstrap-menu" class="navbar navbar-default navbar-fixed-top" role="navigation">
        <div class="container">
+
    <div class="container-fluid">
        <nav class="navbar transparent navbar-default navbar-fixed-top">
+
        <div class="navbar-header">
          <div class="container">
+
            <button type="button" class="navbar-toggle" data-toggle="collapse" data-target=".navbar-menubuilder"><span class="sr-only">Toggle navigation</span><span class="icon-bar"></span><span class="icon-bar"></span><span class="icon-bar"></span>
            <div class="navbar-header">
+
            </button>
              <button type="button" class="navbar-toggle collapsed" data-toggle="collapse" data-target="#navbar" aria-expanded="false" aria-controls="navbar">
+
        </div>
                <span class="sr-only">Toggle navigation</span>
+
        <div class="collapse navbar-collapse navbar-menubuilder">
                <span class="icon-bar"></span>
+
            <ul class="nav navbar-nav navbar-right">
                <span class="icon-bar"></span>
+
              <li class="active">
                <span class="icon-bar"></span>
+
              </button>
+
            </div>
+
            <div id="navbar" class="navbar-collapse collapse">
+
              <ul class="nav navbar-nav">
+
                <li class="active">
+
 
                   <a href="https://2015.igem.org/Team:Edinburgh">Home</a></li>
 
                   <a href="https://2015.igem.org/Team:Edinburgh">Home</a></li>
 +
                  <li class="dropdown">
 +
                    <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">About<span class="caret"></span></a>
 +
                    <ul class="dropdown-menu" role="menu">
 +
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Team">Team</a></li>
 +
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Attributions">Attributions</a></li>
 +
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Collaborations">Collaboration</a></li>
 +
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Sponsors">Sponsors</a></li>
 +
 +
                    </ul>
 +
                  </li>
 
                   <li class="dropdown">
 
                   <li class="dropdown">
 
                     <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Project<span class="caret"></span></a>
 
                     <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Project<span class="caret"></span></a>
 
                     <ul class="dropdown-menu" role="menu">
 
                     <ul class="dropdown-menu" role="menu">
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Description">Description</a></li>
+
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Description">Overview</a></li>
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Experiments">Experiments</a> </li>
+
                    <!-- <li><a href="https://2015.igem.org/Team:Edinburgh/Experiments">Experiments</a> </li> -->
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Project/Protocols">Protocols</a> </li>
+
                       <li><a href="https://2015.igem.org/Team:Edinburgh/HeroinBiosensor">Heroin Biosensor</a></li>
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Results">Results</a></li>
+
                      <li><a href="https://2015.igem.org/Team:Edinburgh/DNPBiosensor">DNP Biosensor</a></li>
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Design">Design</a></li>
+
                       <li><a href="https://2015.igem.org/Team:Edinburgh/PMABiosensor">PMA Biosensor</a></li>
 +
                       <li><a href="https://2015.igem.org/Team:Edinburgh/CBD">Making it Stick</a></li>           
 +
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Results">Limits of Detection</a></li>
 
                     </ul>
 
                     </ul>
 
                   </li>
 
                   </li>
Line 40: Line 43:
 
                     <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Parts<span class="caret"></span></a>
 
                     <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Parts<span class="caret"></span></a>
 
                     <ul class="dropdown-menu" role="menu">
 
                     <ul class="dropdown-menu" role="menu">
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Parts">Team Parts</a></li>
+
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Parts">Team Parts</a></li>  
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Basic_Part">Basic Parts</a></li>
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Basic_Part">Basic Parts</a></li>
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Composite_Part">Composite Parts</a></li>
 
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Composite_Part">Composite Parts</a></li>
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Part_Collection">Part Collection</a> </li>
+
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Part_Collection">Part Collection</a> </li>
 +
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Improved_Part">Improved Parts</a></li>
 +
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Characterisation_Part">Improved Characterisation</a></li>
 +
                    </ul>
 +
                  </li>
 +
                  <li class="dropdown">
 +
                    <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Dry Lab<span class="caret"></span></a>
 +
                    <ul class="dropdown-menu" role="menu">
 +
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Modeling">Modeling</a></li>
 +
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Design">Design</a></li>
 +
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Software">Software</a></li>
 +
                    </ul>
 +
                  </li>
 +
                  <li class="dropdown">
 +
                    <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Human Practices<span class="caret"></span></a>
 +
                    <ul class="dropdown-menu" role="menu">
 +
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Practices">Our Story</a></li>
 +
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Safety">Safety</a> </li>
 +
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Legality">Legality</a> </li>
 +
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Outreach">Outreach</a> </li>
 
                     </ul>
 
                     </ul>
 
                   </li>
 
                   </li>
                  <li><a href="https://2015.igem.org/Team:Edinburgh/Attributions">Attributions</a></li>
 
                  <li><a href="https://2015.igem.org/Team:Edinburgh/Practices">Policy and Practices</a></li>
 
                  <li><a href="https://2015.igem.org/Team:Edinburgh/Safety">Safety</a></li>
 
                  <li><a href="https://2015.igem.org/Team:Edinburgh/Modeling">Modeling</a></li>
 
 
                   <li class="dropdown">
 
                   <li class="dropdown">
 
                     <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">InterLab<span class="caret"></span></a>
 
                     <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">InterLab<span class="caret"></span></a>
Line 57: Line 75:
 
                     </ul>
 
                     </ul>
 
                   </li>
 
                   </li>
                   <li><a href="software.html">Software</a></li>
+
                   <li class="dropdown">
                  <li><a href="entrepreneurship.html">Entrepreneurship</a></li>
+
                    <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Notebook<span class="caret"></span></a>
                  <li><a href="collaborations.html">Collaborations</a></li>
+
                    <ul class="dropdown-menu" role="menu">
                </ul>
+
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Project/Protocols">Protocols</a></li>
              </div>
+
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Notebook/HeroinPurity">Heroin Purity</a></li>
            </div>
+
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Notebook/PMADetection">PMA Detection</a> </li>
          </nav>
+
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Notebook/DNPDetection">DNP Detection</a> </li>
 +
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Notebook/FluidDynamics">Fluid Dynamics</a> </li>
 +
                    </ul>
 +
                  </li>
 +
                  <li><a href="https://2015.igem.org/Team:Edinburgh/MedalCriteria">Accomplishments</a></li> 
 +
            </ul>
 
         </div>
 
         </div>
      </div>
+
    </div>
 +
</div>
 +
 
 +
 
 
     <!-- End of menu  -->
 
     <!-- End of menu  -->
  
   
+
<body>
 +
   
  
 
       <header class="intro">
 
       <header class="intro">
Line 78: Line 105:
 
                 <p class="intro-text">
 
                 <p class="intro-text">
 
                 </p>
 
                 </p>
 +
                <div align="center">
 +
                    <a href="#about">
 +
                        <span class="arrowtext">Scroll down to read more</span>
 +
                        <img src="https://static.igem.org/mediawiki/2014/3/3e/Aalto_Helsinki_Nuoli.png" class="arrow">
 +
                    </a>
 +
                </div>
 
               </div>
 
               </div>
 
             </div>
 
             </div>
Line 84: Line 117:
 
       </header>
 
       </header>
  
<section id="about">    
+
 
 +
    <section id="about">
 
         <div class="container">
 
         <div class="container">
 
             <div class="row">
 
             <div class="row">
 
                 <div class="col-lg-12 text-center">
 
                 <div class="col-lg-12 text-center">
                     <h2 class="section-heading">OVERVIEW</h2>
+
                     <h2 class="section-heading">Overview</h2>
 
                     <h3 class="section-subheading text-muted">
 
                     <h3 class="section-subheading text-muted">
                    Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean posuere mattis risus ut sollicitudin. Mauris tempus et neque in maximus. Maecenas in velit nec lacus molestie ultrices. Morbi lacinia pellentesque porttitor. Nam nulla enim, congue vel nisl vel, lobortis tempus turpis. Sed semper quis odio et eleifend. Fusce eleifend tortor nec facilisis finibus. Sed mollis erat et sapien convallis, posuere cursus lectus iaculis. Pellentesque at augue varius, molestie nulla vel, fringilla nisl. Aenean id rhoncus ante. Pellentesque at urna sed tortor porttitor scelerisque. Mauris lacinia ligula maximus, pulvinar dolor vel, varius nisl. Suspendisse pellentesque augue ac ornare tempor. Praesent sit amet purus at dolor aliquam molestie nec vitae ipsumLorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean posuere mattis risus ut sollicitudin. Mauris tempus et neque in maximus. Maecenas in velit nec lacus molestie ultrices. Morbi lacinia pellentesque porttitor. Nam nulla enim, congue vel nisl vel, lobortis tempus turpis. Sed semper quis odio et eleifend. Fusce eleifend tortor nec facilisis finibus. Sed mollis erat et sapien convallis, posuere cursus lectus iaculis. Pellentesque at augue varius, molestie nulla vel, fringilla nisl. Aenean id rhoncus ante. Pellentesque at urna sed tortor porttitor scelerisque. Mauris lacinia ligula maximus, pulvinar dolor vel, varius nisl. Suspendisse pellentesque augue ac ornare tempor. Praesent sit amet purus at dolor aliquam molestie nec vitae ipsum
+
                      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).  
 +
<br>
 +
<br>
 +
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.
 +
<br>
 +
<br>
 +
More detailed information regarding the cloning process can be found below and on our InterLab lab notebook page.
 
                     </h3>
 
                     </h3>
 
                 </div>
 
                 </div>
 
             </div>
 
             </div>
 +
 +
 +
<section id="about">
 +
    <div class="container">
 +
            <div class="row">
 +
                <div class="col-lg-12 text-center">
 +
                    <h2 class="section-heading">Protocols</h2>
 +
                    <h3 class="section-subheading text-muted">
 +
                      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.
 +
                    </h3>                 
 +
                </div>
 +
            </div>
 +
  </div>
 +
</section>
 +
 +
<div class="container">
 +
 +
    <div id="accordion" class="panel-group">
 +
 +
        <div class="panel panel-default">
 +
 +
            <div class="panel-heading">
 +
 +
                <h4 class="panel-title">
 +
 +
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseOne">What protocol was followed for growing cells for measurement?</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseOne" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
 +
                    <p>The protocol supplied in the iGEM 2015 InterLab Protocol  form was followed with a few minor changes.
 +
                    <br>
 +
                    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>
 +
 +
                </div>
 +
 +
            </div>
 +
 +
        </div>
 +
 +
        <div class="panel panel-default">
 +
 +
            <div class="panel-heading">
 +
 +
                <h4 class="panel-title">
 +
 +
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwo">What controls were used?</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseTwo" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
 +
                    <p>The positive control used was the recommended BBa_I20270, a chloramphenicol resistant GFP expression device in pSB1C3.
 +
<br>
 +
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>
 +
 +
                </div>
 +
 +
            </div>
 +
 +
        </div>
 +
 +
        <div class="panel panel-default">
 +
 +
            <div class="panel-heading">
 +
 +
                <h4 class="panel-title">
 +
 +
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseThree">What agar was used to grow the cells?</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseThree" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
 +
                    <p>The cells were grown on LB agar supplemented with 1000x chloramphenicol.</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="#collapseFour">How were the cells prepared for measurement?</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseFour" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
 +
                    <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.
 +
<br>
 +
For each device three colonies were inoculated to act as the three biological replicates.</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="#collapseFive">How was the fluorescence of each device measured? (followed protocol)</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseFive" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
 +
                    <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.
 +
<br>
 +
To measure fluorescence, the cells were excited at a wavelength of 485nm and emitted light at 538nm as measured.
 +
<br>
 +
The values obtained for fluorescence were in relative fluorescence units.</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="#collapseSix">What instrument was used to measure the fluorescence of each device?</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseSix" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
 +
                    <p>A SpectaMax M5 plate reader was used to obtain the values of fluorescence.</p>
 +
 +
                </div>
 +
 +
            </div>
 +
 +
        </div>
 +
 +
    </div>
 
</div>
 
</div>
  
 +
<section id="about">
 +
    <div class="container">
 +
            <div class="row">
 +
                <div class="col-lg-12 text-center">
 +
                    <h2 class="section-heading">Results</h2>                   
 +
                </div>
 +
            </div>
 +
  </div>
 
</section>
 
</section>
  
<div class="container">  
+
<div class="container">
     <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
+
 
      <div class="panel panel-default">
+
     <div id="accordion" class="panel-group">
        <div class="panel-heading" role="tab" id="headingOne">
+
 
          <h4 class="panel-title">
+
        <div class="panel panel-default">
            <a role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseOne" aria-expanded="false" aria-controls="collapseOne">
+
 
              SECTION I - LOREM IPSUM
+
            <div class="panel-heading">
            </a>
+
 
          </h4>
+
                <h4 class="panel-title">
        <h6> Neque porro quisquam est qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit.</h6>
+
 
 +
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseSeven">How was the raw data processed?</a>
 +
 
 +
                </h4>
 +
 
 +
            </div>
 +
 
 +
            <div id="collapseSeven" class="panel-collapse collapse">
 +
 
 +
                <div class="panel-body">
 +
 
 +
                    <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>
 +
 
 +
                </div>
 +
 
 +
            </div>
 +
 
 
         </div>
 
         </div>
         <div id="collapseOne" class="panel-collapse collapse in" role="tabpanel" aria-labelledby="headingOne">
+
 
          <div class="panel-body">
+
         <div class="panel panel-default">
            <p>
+
 
                <h5>Lorem ipsum dolor sit amet?</h5>
+
            <div class="panel-heading">
                Lorem ipsum dolor sit amet.Lorem ipsum dolor sit amet.Lorem ipsum dolor sit amet.
+
 
            </p>
+
                <h4 class="panel-title">
          </div>
+
 
        <div class="panel-body">
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseEight">What were the units of the results?</a>
            <p>
+
 
                <h5>Lorem ipsum dolor sit amet?</h5>
+
                </h4>
                Lorem ipsum dolor sit amet.Lorem ipsum dolor sit amet.Lorem ipsum dolor sit amet.
+
 
            </p>
+
            </div>
          </div>
+
 
 +
            <div id="collapseEight" class="panel-collapse collapse">
 +
 
 +
                <div class="panel-body">
 +
 
 +
                    <p>The data is in relative fluorescent units.</p>
 +
                </div>
 +
 
 +
            </div>
 +
 
 
         </div>
 
         </div>
      </div>
 
  
 +
        <div class="panel panel-default">
 +
 +
            <div class="panel-heading">
 +
 +
                <h4 class="panel-title">
 +
 +
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseNine">Fluorescence of 3 biological replicates of positive control</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseNine" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
<p>1.) 1278.712 <br>  2.) 1216.887  <br>  3.) 1110.834</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="#collapseTen">Fluorescence of 3 biological replicates of negative control</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseTen" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
 +
                    <p>1.) -36.863 <br>  2.) -33.066 <br>    3.) -44.696</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="#collapseEleven">Fluorescence of 3 biological replicates of J23101 + I13504, mean and st.dev (device 1)</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseEleven" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
 +
                    <p>1.)5082.078 <br> 2.) 5879.239 <br> 3.) 5973.973
 +
                        <br>
 +
                        Mean = 5645.097
 +
                        <br>
 +
                        Standard Deviation = 489.884
 +
                  </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="#collapseTwelve">Fluorescence of 3 biological replicates of J23106 + I13504, mean and st.dev (device 2)</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseTwelve" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
 +
                    <p>
 +
                      1.) 3185.188 <br> 2.) 3119.913 <br> 3.) 2396.115
 +
                      <br>
 +
                      Mean = 2900.405
 +
                      <br>
 +
                      Standard Deviation = 437.946
 +
                  </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="#collapseThirteen">Fluorescence of 3 biological replicates of J23117 + I13504, mean and st.dev (device3)</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseThirteen" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
 +
                    <p>
 +
                      1.) -14.003 <br> 2.) -27.149 <br> 3.) -16.631
 +
                      <br>
 +
                      Mean = -19.261
 +
                      <br>
 +
                      Standard Deviation = 6.956
 +
                  </p>
 +
 +
                </div>
 +
 +
            </div>
 +
 +
        </div>
 +
    </div>
 +
</div>
 +
 +
<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>
 +
  </div>
 +
</section>
 +
 +
<div class="container">
 +
 +
    <div id="accordion" class="panel-group">
 +
 +
        <div class="panel panel-default">
 +
 +
            <div class="panel-heading">
 +
 +
                <h4 class="panel-title">
 +
 +
                    <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>
 +
 +
            </div>
 +
 +
            <div id="collapseFourteen" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
 +
                    <p>2nd Replicate = 5202.079 <br> 3rd replicate = 5187.619</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="#collapseFifteen">Fluorescence of 2nd and 3rd technical replicates of J23101 + I13504 (device 1) biological replicate 2</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseFifteen" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
 +
                    <p>2nd replicate = 6008.646 <br> 3rd replicate = 6135.483</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="#collapseSixteen">Fluorescence of 2nd and 3rd technical replicates of J23101 + I13504 (device 1) biological replicate 3</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseSixteen" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
 +
                    <p>2nd replicate =6634.865 <br> 3rd replicate = 6254.715</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="#collapseSeventeen">Mean and standard deviation of technical replicates of J23101 + I13504 (device 1)</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseSeventeen" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
 +
                    <p>
 +
Mean = 5817.633
 
<br>
 
<br>
      <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
+
Standard deviation = 541.009
      <div class="panel panel-default">
+
</p>
        <div class="panel-heading" role="tab" id="headingOne">
+
 
          <h4 class="panel-title">
+
                </div>
            <a role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseOne" aria-expanded="false" aria-controls="collapseOne">
+
 
              SECTION II - LOREM IPSUM
+
             </div>
             </a>
+
 
          </h4>
+
        <h6> Neque porro quisquam est qui dolorem ipsum quia dolor sit amet, consectetur, adipisci velit.</h6>
+
 
         </div>
 
         </div>
         <div id="collapseOne" class="panel-collapse collapse in" role="tabpanel" aria-labelledby="headingOne">
+
          
          <div class="panel-body">
+
                <div class="panel panel-default">
            <p>
+
 
                <h5>Lorem ipsum dolor sit amet?</h5>
+
            <div class="panel-heading">
                Lorem ipsum dolor sit amet.Lorem ipsum dolor sit amet.Lorem ipsum dolor sit amet.
+
 
             </p>
+
                <h4 class="panel-title">
          </div>
+
 
        <div class="panel-body">
+
                    <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>
            <p>
+
 
                <h5>Lorem ipsum dolor sit amet?</h5>
+
                </h4>
                Lorem ipsum dolor sit amet.Lorem ipsum dolor sit amet.Lorem ipsum dolor sit amet.
+
 
            </p>
+
             </div>
          </div>
+
 
 +
            <div id="collapseEighteen" class="panel-collapse collapse">
 +
 
 +
                <div class="panel-body">
 +
 
 +
                    <p>2nd replicate = 3694.877 <br> 3rd replicate = 3606.107</p>
 +
 
 +
                </div>
 +
 
 +
            </div>
 +
 
 
         </div>
 
         </div>
      </div>
 
  
 +
                <div class="panel panel-default">
  
 +
            <div class="panel-heading">
  
 +
                <h4 class="panel-title">
  
 +
                    <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>
  
 +
            <div id="collapseNineteen" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
 +
                    <p>2nd replicate = 3036.241 <br> 3rd replicate = 3140.1</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="#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">
 +
 +
                <div class="panel-body">
 +
 +
                    <p>2nd replicate = 2871.642 <br> 3rd replicate = 2801.428</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="#collapseTwentyOne">Mean and standard deviation of technical replicates of J23106 + I13504 (device 2)</a>
 +
 +
                </h4>
 +
 +
            </div>
 +
 +
            <div id="collapseTwentyOne" class="panel-collapse collapse">
 +
 +
                <div class="panel-body">
 +
 +
                    <p>Mean = 3094.623
 +
<br>
 +
Standard deviation = 396.837</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="#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">
 +
 +
                <div class="panel-body">
 +
 +
                    <p>2nd replicate = 7.859 <br> 3rd replicate = 0.659</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="#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>
 +
 +
    <div class="panel panel-default">
 +
 +
            <div class="panel-heading">
 +
 +
                <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>
  
  

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