Difference between revisions of "Team:Edinburgh/Composite Part"

 
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{{Edinburgh_practices}}
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{{Edinburgh_Composite}}
 
<html>
 
<html>
  
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<script src="https://maxcdn.bootstrapcdn.com/bootstrap/3.3.5/js/bootstrap.min.js"></script>
 
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                   <a href="https://2015.igem.org/Team:Edinburgh">Home</a></li>
 
                   <a href="https://2015.igem.org/Team:Edinburgh">Home</a></li>
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                    <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">About<span class="caret"></span></a>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Team">Team</a></li>
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                       <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>
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                    <!-- <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>
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                       <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>
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                     <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>
 
                     </ul>
 
                   </li>
 
                   </li>
                  <li><a href="https://2015.igem.org/Team:Edinburgh/Attributions">Attributions</a></li>
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                  <li class="dropdown">
                  <li><a href="https://2015.igem.org/Team:Edinburgh/Practices">Policy and Practices</a></li>
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                    <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Dry Lab<span class="caret"></span></a>
                  <li><a href="https://2015.igem.org/Team:Edinburgh/Safety">Safety</a></li>
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                    <ul class="dropdown-menu" role="menu">
                  <li><a href="https://2015.igem.org/Team:Edinburgh/Modeling">Modeling</a></li>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Modeling">Modeling</a></li>
                   <li><a href="https://2015.igem.org/Team:Edinburgh/InterLab">InterLab</a></li>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Design">Design</a></li>
                  <li><a href="software.html">Software</a></li>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Software">Software</a></li>
                   <li><a href="entrepreneurship.html">Entrepreneurship</a></li>
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                    </ul>
                  <li><a href="collaborations.html">Collaborations</a></li>
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                  </li>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Practices">Our Story</a></li>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Safety">Safety</a> </li>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Outreach">Outreach</a> </li>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/InterLab">InterLab</a></li>
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                    <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Notebook<span class="caret"></span></a>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Project/Protocols">Protocols</a></li>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Notebook/HeroinPurity">Heroin Purity</a></li>
 +
                      <li><a href="https://2015.igem.org/Team:Edinburgh/Notebook/PMADetection">PMA Detection</a> </li>
 +
                      <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>
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    </div>
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</div>
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     <!-- End of menu  -->
 
     <!-- End of menu  -->
  
        <header class="intro">
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<body>
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 +
          <header class="intro">
 
         <div class="intro-body">
 
         <div class="intro-body">
 
           <div class="container">
 
           <div class="container">
 
             <div class="row">
 
             <div class="row">
 
               <div class="col-md-12">
 
               <div class="col-md-12">
                 <h1 class="brand-heading">Basic Parts</h1>
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                 <h1 class="brand-heading">Composite Parts</h1>
 
                 <p class="intro-text">
 
                 <p class="intro-text">
<br>
 
<br>
 
<br>
 
 
                 </p>
 
                 </p>
 +
                <div align="center">
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                    <a href="#cd-team">
 +
                        <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">
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                    </a>
 +
                </div>
 
               </div>
 
               </div>
 
             </div>
 
             </div>
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  <div class="container">  
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<section id="about">
<div class="panel panel-default">
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    <div class="container">
  <div class="panel-heading">
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            <div class="row">
    <h3 class="panel-title">Panel title</h3>
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                <div class="col-lg-12 text-center">
  </div>
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                    <h2 class="section-heading">mRFP</h2>                  
</div>
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                 </div>
    <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
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      <div class="panel panel-default">
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        <div class="panel-heading" role="tab" id="headingOne">
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          <h4 class="panel-title">
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            <a role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseOne" aria-expanded="false" aria-controls="collapseOne">
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              1% Agarose Gel
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            </a>
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          </h4>
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        </div>
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        <div id="collapseOne" class="panel-collapse collapse in" role="tabpanel" aria-labelledby="headingOne">
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          <div class="panel-body">
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            <div class="col-md-6">
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              <p style="color: black;">
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              <h2>Materials</h2>
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              <ul>
+
                 <li>1g Agarose
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                <li>100ml 1X TAE buffer
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                <li>5µl GelRed stain
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              </ul>
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            </p>
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             </div>
 
             </div>
            <div class="col-md-6">
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  </div>
              <p class="text-muted">
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</section>
              <h2>Procedure</h2>
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              <ul>
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<div class="container">
                <li>1. Mix the agarose with the 1X TAE buffer in a flask.
+
 
                <li>2. Heat the mixture until all the agarose is dissolved.
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    <div id="accordion" class="panel-group">
                <li>3. Swirl the flask under cold running water to cool the mixture.
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                 <li> 4. Add the gel stain.
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        <div class="panel panel-default">
                <li>5. Pour into an assembled gel tray and let it cool.
+
 
              </uL>
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            <div class="panel-heading">
            </p>
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 +
                 <h4 class="panel-title">
 +
 
 +
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseOne">mRFP driven by LacI promoter (BBa_K1615090)</a>
 +
 
 +
                </h4>
 +
 
 
             </div>
 
             </div>
          </div>
 
        </div>
 
      </div>
 
      <div class="panel panel-default">
 
        <div class="panel-heading" role="tab" id="headingTwo">
 
          <h4 class="panel-title">
 
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseTwo" aria-expanded="false" aria-controls="collapseTwo">
 
            Agarose Gel Electrophoresis
 
            </a>
 
          </h4>
 
        </div>
 
        <div id="collapseTwo" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingTwo">
 
          <div class="panel-body">
 
            <div class="col-md-6">
 
              <p>
 
              <h2>Materials</h2>
 
              <ul>
 
                <li>1% Agarose
 
                <li>1X TAE buffer
 
                <li>5X loading dye
 
                <li>DNA ladder
 
                <li>DNA samples
 
              </ul>
 
            </p>
 
            </div>
 
            <div class="col-md-6">
 
              <p>
 
              <h2>Procedure</h2>
 
              <ul>
 
                <li>1. Place gel tray into the electrophoresis apparatus.
 
                <li>2. Pour 1X TAE so that the gel is covered by buffer.
 
                <li>3. Prepare the samples by adding the appropriate amount of loading dye.
 
                <li>4. Load samples and DNA ladder into wells on the gel.
 
                <li>5. Run the gel at roughly 100V for around an hour
 
  
              </uL>
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            <div id="collapseOne" class="panel-collapse collapse">
            </p>
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 +
                <div class="panel-body">
 +
 
 +
                    <p>mRFP (BBa_K1615089) driven by the IPTG inducible PLlac 0-1 promoter (BBa_K314103). This allows the <i>mRFP</i> gene to express mRFP when induced with IPTG.
 +
</p>
 +
 
 +
                </div>
 +
 
 
             </div>
 
             </div>
          </div>
+
 
 
         </div>
 
         </div>
      </div>
+
 
      <div class="panel panel-default">
+
        <div class="panel panel-default">
        <div class="panel-heading" role="tab" id="headingThree">
+
 
          <h4 class="panel-title">
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            <div class="panel-heading">
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseThree" aria-expanded="false" aria-controls="collapseThree">
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              Culture
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                <h4 class="panel-title">
            </a>
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          </h4>
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwo">CBDcenA+Linker fused to mRFP in RFC25 (BBa_K1615091)</a>
        </div>
+
 
        <div id="collapseThree" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingThree">
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                 </h4>
          <div class="panel-body">
+
 
          <div class="col-md-6">
+
              <p>
+
              <h2>Materials</h2>
+
              <ul>
+
                 <li>10ml Luria Broth (LB)
+
                <li>10µl Specific Antibiotic at 1000x (Chloramphenicol, Ampicillin or Kanamycin)
+
                <li>Loop (for picking colony)
+
                <li>Ethanol
+
              </ul>
+
            </p>
+
 
             </div>
 
             </div>
             <div class="col-md-6">
+
 
              <p>
+
             <div id="collapseTwo" class="panel-collapse collapse">
              <h2>Procedure</h2>
+
 
              <ul>
+
                <div class="panel-body">
                <li>1. Pour 10ml of LB into a 50ml Falcon tube.  
+
 
                <li>2. Pipette 10µl of antibiotic into the broth.
+
                    <p>mRFP (BBa_K1615089) fused to the C terminal of CBDcenA + C-terminal linker (BBa_K1321339) in RFC25. If folded properly, this allows mRFP to fluoresce while immobilised on cellulose.
                <li>3. Dip loop in ethanol and flame to sterilise. Once it is cool, pick colony and transfer to a 50ml Falcon tube.
+
<br>
                <li>4. Incubate at 37°C overnight in a shaking incubator.
+
<img src="https://static.igem.org/mediawiki/2015/2/20/CBD_RFP_folding.jpeg">
              </uL>
+
<br>
            </p>
+
To test the RFP folding when fused to a CBD we placed 200µL of crude cell lysate in a 96 well plate and observed the fluorescence under a blue light transilluminator.  
 +
 
 +
</p>
 +
 
 +
                </div>
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             </div>
 
             </div>
          </div>
 
        </div>
 
      </div>
 
    </div>
 
  </div>
 
  
<div class="panel panel-default">
 
  <div class="panel-heading">
 
    <h3 class="panel-title">Panel title</h3>
 
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</div>
 
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      <div class="panel panel-default">
 
        <div class="panel-heading" role="tab" id="headingOne">
 
          <h4 class="panel-title">
 
            <a role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseOne" aria-expanded="false" aria-controls="collapseOne">
 
              1% Agarose Gel
 
            </a>
 
          </h4>
 
 
         </div>
 
         </div>
         <div id="collapseOne" class="panel-collapse collapse in" role="tabpanel" aria-labelledby="headingOne">
+
 
          <div class="panel-body">
+
         <div class="panel panel-default">
            <div class="col-md-6">
+
 
              <p style="color: black;">
+
            <div class="panel-heading">
              <h2>Materials</h2>
+
 
              <ul>
+
                <h4 class="panel-title">
                 <li>1g Agarose
+
 
                <li>100ml 1X TAE buffer
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseFour">mRFP fused to CBDcenA+Linker in RFC25 (BBa_K16150092)</a>
                <li>5µl GelRed stain
+
 
              </ul>
+
                 </h4>
            </p>
+
 
 
             </div>
 
             </div>
            <div class="col-md-6">
 
              <p class="text-muted">
 
              <h2>Procedure</h2>
 
              <ul>
 
                <li>1. Mix the agarose with the 1X TAE buffer in a flask.
 
                <li>2. Heat the mixture until all the agarose is dissolved.
 
                <li>3. Swirl the flask under cold running water to cool the mixture.
 
                <li> 4. Add the gel stain.
 
                <li>5. Pour into an assembled gel tray and let it cool.
 
              </uL>
 
            </p>
 
            </div>
 
          </div>
 
        </div>
 
      </div>
 
      <div class="panel panel-default">
 
        <div class="panel-heading" role="tab" id="headingTwo">
 
          <h4 class="panel-title">
 
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseTwo" aria-expanded="false" aria-controls="collapseTwo">
 
            Agarose Gel Electrophoresis
 
            </a>
 
          </h4>
 
        </div>
 
        <div id="collapseTwo" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingTwo">
 
          <div class="panel-body">
 
            <div class="col-md-6">
 
              <p>
 
              <h2>Materials</h2>
 
              <ul>
 
                <li>1% Agarose
 
                <li>1X TAE buffer
 
                <li>5X loading dye
 
                <li>DNA ladder
 
                <li>DNA samples
 
              </ul>
 
            </p>
 
            </div>
 
            <div class="col-md-6">
 
              <p>
 
              <h2>Procedure</h2>
 
              <ul>
 
                <li>1. Place gel tray into the electrophoresis apparatus.
 
                <li>2. Pour 1X TAE so that the gel is covered by buffer.
 
                <li>3. Prepare the samples by adding the appropriate amount of loading dye.
 
                <li>4. Load samples and DNA ladder into wells on the gel.
 
                <li>5. Run the gel at roughly 100V for around an hour
 
  
              </uL>
+
            <div id="collapseFour" class="panel-collapse collapse">
            </p>
+
 
 +
                <div class="panel-body">
 +
 
 +
                    <p>mRFP (BBa_K1615089) fused to the N terminal of CBDcenA + C terminal linker (BBa_K1321339) in RFC25. If folded properly, this allows mRFP to fluoresce while immobilised on cellulose.
 +
<br>
 +
<br>
 +
<img src="https://static.igem.org/mediawiki/2015/2/20/CBD_RFP_folding.jpeg">
 +
<br>
 +
<br>
 +
We tested the dissociation of this part on Whatman 54 using isostandard chads. This involved incubating the chads in the crude cell lysate for 10 minutes and then washing them with PBS for various periods time to test the binding affinity of this CBD.
 +
<br>
 +
<br>
 +
We tested the dissociation of this part on Whatman 54 using isostandard chads. This involved incubating the chads in the crude cell lysate for 10 minutes and then washing them with PBS for various periods time to test the binding affinity of this CBD.
 +
 
 +
</p>
 +
 
 +
                </div>
 +
 
 
             </div>
 
             </div>
          </div>
 
        </div>
 
      </div>
 
      <div class="panel panel-default">
 
        <div class="panel-heading" role="tab" id="headingThree">
 
          <h4 class="panel-title">
 
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseThree" aria-expanded="false" aria-controls="collapseThree">
 
              Culture
 
            </a>
 
          </h4>
 
        </div>
 
        <div id="collapseThree" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingThree">
 
          <div class="panel-body">
 
          <div class="col-md-6">
 
              <p>
 
              <h2>Materials</h2>
 
              <ul>
 
                <li>10ml Luria Broth (LB)
 
                <li>10µl Specific Antibiotic at 1000x (Chloramphenicol, Ampicillin or Kanamycin)
 
                <li>Loop (for picking colony)
 
                <li>Ethanol
 
              </ul>
 
            </p>
 
            </div>
 
            <div class="col-md-6">
 
              <p>
 
              <h2>Procedure</h2>
 
              <ul>
 
                <li>1. Pour 10ml of LB into a 50ml Falcon tube.
 
                <li>2. Pipette 10µl of antibiotic into the broth.
 
                <li>3. Dip loop in ethanol and flame to sterilise. Once it is cool, pick colony and transfer to a 50ml Falcon tube.
 
                <li>4. Incubate at 37°C overnight in a shaking incubator.
 
              </uL>
 
            </p>
 
            </div>
 
          </div>
 
        </div>
 
      </div>
 
      <div class="panel panel-default">
 
        <div class="panel-heading" role="tab" id="headingFour">
 
          <h4 class="panel-title">
 
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseFour" aria-expanded="false" aria-controls="collapseFour">
 
            Gel Purification using QIAquick Gel Extraction Kit
 
            </a>
 
          </h4>
 
        </div>
 
      </div>
 
    </div>
 
  </div>
 
  
<div class="panel panel-default">
 
  <div class="panel-heading">
 
    <h3 class="panel-title">Panel title</h3>
 
  </div>
 
</div>
 
    <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true">
 
      <div class="panel panel-default">
 
        <div class="panel-heading" role="tab" id="headingOne">
 
          <h4 class="panel-title">
 
            <a role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseOne" aria-expanded="false" aria-controls="collapseOne">
 
              1% Agarose Gel
 
            </a>
 
          </h4>
 
 
         </div>
 
         </div>
        <div id="collapseOne" class="panel-collapse collapse in" role="tabpanel" aria-labelledby="headingOne">
 
          <div class="panel-body">
 
            <div class="col-md-6">
 
              <p style="color: black;">
 
              <h2>Materials</h2>
 
              <ul>
 
                <li>1g Agarose
 
                <li>100ml 1X TAE buffer
 
                <li>5µl GelRed stain
 
              </ul>
 
            </p>
 
            </div>
 
            <div class="col-md-6">
 
              <p class="text-muted">
 
              <h2>Procedure</h2>
 
              <ul>
 
                <li>1. Mix the agarose with the 1X TAE buffer in a flask.
 
                <li>2. Heat the mixture until all the agarose is dissolved.
 
                <li>3. Swirl the flask under cold running water to cool the mixture.
 
                <li> 4. Add the gel stain.
 
                <li>5. Pour into an assembled gel tray and let it cool.
 
              </uL>
 
            </p>
 
            </div>
 
          </div>
 
        </div>
 
      </div>
 
      <div class="panel panel-default">
 
        <div class="panel-heading" role="tab" id="headingTwo">
 
          <h4 class="panel-title">
 
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseTwo" aria-expanded="false" aria-controls="collapseTwo">
 
            Agarose Gel Electrophoresis
 
            </a>
 
          </h4>
 
        </div>
 
        <div id="collapseTwo" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingTwo">
 
          <div class="panel-body">
 
            <div class="col-md-6">
 
              <p>
 
              <h2>Materials</h2>
 
              <ul>
 
                <li>1% Agarose
 
                <li>1X TAE buffer
 
                <li>5X loading dye
 
                <li>DNA ladder
 
                <li>DNA samples
 
              </ul>
 
            </p>
 
            </div>
 
            <div class="col-md-6">
 
              <p>
 
              <h2>Procedure</h2>
 
              <ul>
 
                <li>1. Place gel tray into the electrophoresis apparatus.
 
                <li>2. Pour 1X TAE so that the gel is covered by buffer.
 
                <li>3. Prepare the samples by adding the appropriate amount of loading dye.
 
                <li>4. Load samples and DNA ladder into wells on the gel.
 
                <li>5. Run the gel at roughly 100V for around an hour
 
  
              </uL>
+
        <div class="panel panel-default">
             </p>
+
 
 +
             <div class="panel-heading">
 +
 
 +
                <h4 class="panel-title">
 +
 
 +
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseFive">Linker +dCBD fused to mRFP in RFC25 (BBa_K1615093)
 +
</a>
 +
 
 +
                </h4>
 +
 
 
             </div>
 
             </div>
          </div>
 
        </div>
 
      </div>
 
      <div class="panel panel-default">
 
        <div class="panel-heading" role="tab" id="headingThree">
 
          <h4 class="panel-title">
 
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseThree" aria-expanded="false" aria-controls="collapseThree">
 
              Culture
 
            </a>
 
          </h4>
 
        </div>
 
        <div id="collapseThree" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingThree">
 
          <div class="panel-body">
 
          <div class="col-md-6">
 
              <p>
 
              <h2>Materials</h2>
 
              <ul>
 
                <li>10ml Luria Broth (LB)
 
                <li>10µl Specific Antibiotic at 1000x (Chloramphenicol, Ampicillin or Kanamycin)
 
                <li>Loop (for picking colony)
 
                <li>Ethanol
 
              </ul>
 
            </p>
 
            </div>
 
            <div class="col-md-6">
 
              <p>
 
              <h2>Procedure</h2>
 
              <ul>
 
                <li>1. Pour 10ml of LB into a 50ml Falcon tube.
 
                <li>2. Pipette 10µl of antibiotic into the broth.
 
                <li>3. Dip loop in ethanol and flame to sterilise. Once it is cool, pick colony and transfer to a 50ml Falcon tube.
 
                <li>4. Incubate at 37°C overnight in a shaking incubator.
 
              </uL>
 
            </p>
 
            </div>
 
          </div>
 
        </div>
 
      </div>
 
      <div class="panel panel-default">
 
        <div class="panel-heading" role="tab" id="headingFour">
 
          <h4 class="panel-title">
 
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseFour" aria-expanded="false" aria-controls="collapseFour">
 
            Gel Purification using QIAquick Gel Extraction Kit
 
            </a>
 
          </h4>
 
        </div>
 
      </div>
 
    </div>
 
  </div>
 
</div>
 
  
 +
            <div id="collapseFive" class="panel-collapse collapse">
  
 +
                <div class="panel-body">
  
 +
                    <p>mRFP (BBa_K1615089) fused to the C terminal of dCBD + N terminal linker (BBa_K1321340). If folded properly, this allows mRFP to fluoresce while immobilised on cellulose.
 +
<br>
 +
<br>
 +
<img src="https://static.igem.org/mediawiki/2015/2/20/CBD_RFP_folding.jpeg">
 +
<br>
 +
To test the RFP folding when fused to a CBD we placed 200µL of crude cell lysate in a 96 well plate and observed the fluorescence under a blue light transilluminator.
 +
</p>
  
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Latest revision as of 19:10, 20 November 2015

mRFP

mRFP (BBa_K1615089) driven by the IPTG inducible PLlac 0-1 promoter (BBa_K314103). This allows the mRFP gene to express mRFP when induced with IPTG.

mRFP (BBa_K1615089) fused to the C terminal of CBDcenA + C-terminal linker (BBa_K1321339) in RFC25. If folded properly, this allows mRFP to fluoresce while immobilised on cellulose.

To test the RFP folding when fused to a CBD we placed 200µL of crude cell lysate in a 96 well plate and observed the fluorescence under a blue light transilluminator.

mRFP (BBa_K1615089) fused to the N terminal of CBDcenA + C terminal linker (BBa_K1321339) in RFC25. If folded properly, this allows mRFP to fluoresce while immobilised on cellulose.



We tested the dissociation of this part on Whatman 54 using isostandard chads. This involved incubating the chads in the crude cell lysate for 10 minutes and then washing them with PBS for various periods time to test the binding affinity of this CBD.

We tested the dissociation of this part on Whatman 54 using isostandard chads. This involved incubating the chads in the crude cell lysate for 10 minutes and then washing them with PBS for various periods time to test the binding affinity of this CBD.

mRFP (BBa_K1615089) fused to the C terminal of dCBD + N terminal linker (BBa_K1321340). If folded properly, this allows mRFP to fluoresce while immobilised on cellulose.


To test the RFP folding when fused to a CBD we placed 200µL of crude cell lysate in a 96 well plate and observed the fluorescence under a blue light transilluminator.