Difference between revisions of "Team:GreatBay China/Interlab"

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</head>
 
</head>
 
<body>
 
<body>
  
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   <div class="logo"><img src="https://static.igem.org/mediawiki/2018/1/14/T--GreatBay_China--logo1.jpg" /></div>
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   <div class="logo"><a><img src="https://static.igem.org/mediawiki/2018/1/14/T--GreatBay_China--logo1.jpg" /></a></div>
  
 
   <div class="container">  
 
   <div class="container">  
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         <ul>
 
         <ul>
 
           <li>
 
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            <div class="res" target="_self" id="menuhead">
 +
               
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 +
                <h5> Meow~</h5>
 +
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 +
        </li>
 +
         
 
                   <li>
 
                   <li>
 
             <a href="#"><span>Team</span></a>                     
 
             <a href="#"><span>Team</span></a>                     
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                 <a href="https://2018.igem.org/Team:GreatBay_China/Description">Description</a>  
 
                 <a href="https://2018.igem.org/Team:GreatBay_China/Description">Description</a>  
 
                 <a href="https://2018.igem.org/Team:GreatBay_China/Design">Design</a>  
 
                 <a href="https://2018.igem.org/Team:GreatBay_China/Design">Design</a>  
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                 <a href="https://2018.igem.org/Team:GreatBay_China/Experiments">Experiments</a>
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                 <a href="https://2018.igem.org/Team:GreatBay_China/Demonstrate">Results</a>  
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                <a href="https://2018.igem.org/Team:GreatBay_China/Results" >Results</a>  
+
               
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                <a href="https://2018.igem.org/Team:GreatBay_China/Demonstrate">Demonstrate</a>
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                <a href="https://2018.igem.org/Team:GreatBay_China/Improve" >Improve</a>
+
 
           <img src="https://static.igem.org/mediawiki/2018/2/2e/T--GreatBay_China--AlexisWrite.jpgleaf.png"/>
 
           <img src="https://static.igem.org/mediawiki/2018/2/2e/T--GreatBay_China--AlexisWrite.jpgleaf.png"/>
 
          
 
          
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           </li>
 
           </li>
 
           <li>
 
           <li>
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             <a href="https://2018.igem.org/Team:GreatBay_China/Parts"><span>Parts</span></a>  
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             <a href="#"><span>Parts</span></a>  
 
               <div>
 
               <div>
 +
             
 +
              <a href="https://2018.igem.org/Team:GreatBay_China/Parts">Parts</a>
 
                 <a href="https://2018.igem.org/Team:GreatBay_China/Basic_Part">Basic</a>  
 
                 <a href="https://2018.igem.org/Team:GreatBay_China/Basic_Part">Basic</a>  
 
                 <a href="https://2018.igem.org/Team:GreatBay_China/Composite_Part">Composite</a>   
 
                 <a href="https://2018.igem.org/Team:GreatBay_China/Composite_Part">Composite</a>   
−
                 <a href="https://2018.igem.org/Team:GreatBay_China/Part_Collection">Collection</a>  
+
                 <a href="https://2018.igem.org/Team:GreatBay_China/Part_Collection">Collection</a>
 +
                  <a href="https://2018.igem.org/Team:GreatBay_China/Improve" >Improve</a> 
 
           <img src="https://static.igem.org/mediawiki/2018/2/2e/T--GreatBay_China--AlexisWrite.jpgleaf.png"/>
 
           <img src="https://static.igem.org/mediawiki/2018/2/2e/T--GreatBay_China--AlexisWrite.jpgleaf.png"/>
 
          
 
          
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           </li>
 
           </li>
 
           <li>
 
           <li>
−
             <a href="https://2018.igem.org/Team:GreatBay_China/Human_Practices"><span>HP</span></a>
+
             <a href="#"><span>HP</span></a>
 
               <div>  
 
               <div>  
−
                <a href="https://2018.igem.org/Team:GreatBay_China/Engagement">Public <br>Engagement</a>  
+
              <a href="https://2018.igem.org/Team:GreatBay_China/HP">Background</a>  
−
                 <a href="https://2018.igem.org/Team:GreatBay_China/Applied_Design">Product   Design</a>  
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                <a class="long" href="https://2018.igem.org/Team:GreatBay_China/Human_Practices">Integrated HP</a>
−
                 <a href="https://2018.igem.org/Team:GreatBay_China/HP/Gold_Integrated">Integrated HP</a>  
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                 <a class="long" href="https://2018.igem.org/Team:GreatBay_China/Applied_Design">Product Design</a>  
 +
                 <a href="https://2018.igem.org/Team:GreatBay_China/Hardware">Hardware</a>
 +
                <a class="long" href="https://2018.igem.org/Team:GreatBay_China/Public_Engagement">Public Engagement</a>  
 +
               
 +
               
 +
                 
 
           <img src="https://static.igem.org/mediawiki/2018/2/2e/T--GreatBay_China--AlexisWrite.jpgleaf.png"/>
 
           <img src="https://static.igem.org/mediawiki/2018/2/2e/T--GreatBay_China--AlexisWrite.jpgleaf.png"/>
 
          
 
          
 
         </div>
 
         </div>
 
           </li>
 
           </li>
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           <li>
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           <li id="active">
 
             <a href="#"><span>Lab</span></a>
 
             <a href="#"><span>Lab</span></a>
 
               <div>
 
               <div>
 +
                <a href="https://2018.igem.org/Team:GreatBay_China/Experiments">Experiments</a>
 
                 <a href="https://2018.igem.org/Team:GreatBay_China/Notebook">Notebook</a>  
 
                 <a href="https://2018.igem.org/Team:GreatBay_China/Notebook">Notebook</a>  
 
                 <a href="https://2018.igem.org/Team:GreatBay_China/Interlab">Interlab</a>  
 
                 <a href="https://2018.igem.org/Team:GreatBay_China/Interlab">Interlab</a>  
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               <div>
 
               <div>
 
               </div>
 
               </div>
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           <li id="active">
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           <li>
 
             <a href="https://2018.igem.org/Team:GreatBay_China"><span>Home</span></a>
 
             <a href="https://2018.igem.org/Team:GreatBay_China"><span>Home</span></a>
 
           </li>   
 
           </li>   
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     </div>
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       <div class="container">
 
       <div class="container">
 
       <div class="footer">
 
       <div class="footer">
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         <br>
 
         <div class="row" style="padding-left:50px">
 
         <div class="row" style="padding-left:50px">
 +
 
           <div class="contact">
 
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           <img src="https://static.igem.org/mediawiki/2018/d/dd/T--GreatBay_China--footer1.png" width="50px">
 
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           </div>
 
           </div>
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           <h5 style="color:white;">mCATNIP2018@outlook.com</h5>
 
           <h5 style="color:white;">mCATNIP2018@outlook.com</h5>
 +
          </div>
 +
          <div class="contact">
 +
          <img src="https://static.igem.org/mediawiki/2018/e/ed/T--GreatBay_China--footer10.jpeg" width="80px">
 
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           </div>
 
           <div class="contact" >
 
           <div class="contact" >
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           <img src="https://static.igem.org/mediawiki/2018/b/b6/T--GreatBay_China--footer2.png" width="50px">
 
           <img src="https://static.igem.org/mediawiki/2018/b/b6/T--GreatBay_China--footer2.png" width="50px">
 
           </div>
 
           </div>
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           <div class="contact" style="width:451px">
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           <div class="contact" style="width:450px">
 
           <h5 style="color:white;">@Igem2018C</h5>
 
           <h5 style="color:white;">@Igem2018C</h5>
 
           </div>
 
           </div>
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           </div>
 
           </div>
 
           <div class="contact" >
 
           <div class="contact" >
−
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           </div>
 
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       <div class="row">
 
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         <div class="header" style="margin-left: -14px">
 
           <img src="https://static.igem.org/mediawiki/2018/7/74/T--GreatBay_China--Top.png" />
 
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         </div>
 
       </div>
 
       </div>
−
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       <div class="row" style="padding-left:50px">
 
           <h2>Introduction of Interlab</h2>
 
           <h2>Introduction of Interlab</h2>
−
           <h4>   
+
           <h4 style="padding-left:50px;padding-right:50px;">   
−
             Reliable and repeatable measurement play an important role in synthetic biology as an engineering discipline. The InterLab Study aims to identify and correct the sources of systematic variability in synthetic biology measurement, then improve the measurement tools and methods which could enable labs to reliably build upon others’ work. It will promote the collaboration within the synthetic biology community. Enhance, that may promote the synthetic biology to achieve its full potential as an engineering discipline.  
+
             Reliable and repeatable measurement plays an important role as an engineering discipline in synthetic biology. The InterLab Study aims to identify and correct the sources of systematic variability in synthetic biology measurement, thus to improve the measurement tools and methods which can enable labs to reliably build upon others’ work. It will promote the collaboration within the synthetic biology community. Hence, it may propel the synthetic biology to achieve its full potential.
−
             <br>
+
             <br><br>
 
             The previous InterLab studies showed that measuring GFP expression in absolute fluorescence units calibrated against a known concentration of fluorescent molecule can greatly reduce the variability in different labs’ measurements.  
 
             The previous InterLab studies showed that measuring GFP expression in absolute fluorescence units calibrated against a known concentration of fluorescent molecule can greatly reduce the variability in different labs’ measurements.  
−
             <br>
+
             <br><br>
 
             The Fifth Year InterLab’s goal is to remove another source of variability, the number of cells in the sample, by using new cell-counting method.
 
             The Fifth Year InterLab’s goal is to remove another source of variability, the number of cells in the sample, by using new cell-counting method.
  
 
           </h4>  
 
           </h4>  
 
         </div>
 
         </div>
−
<!--DON'T FORGET TEMPLATE STARTS HERE BY CHARLES WEI-->
+
        <br>
 +
      <div class="row" style="padding-left:50px;padding-right:50px;">
 +
          <h4> GreatBay_China investigation had helped in answering the question:<em style="color:red;">Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?</em></h4>
 +
 
 +
      </div><br><br><br>
 +
      <div class="row" style="padding-left:50px">
 +
      <h2> Supplies and Reagents </h2><br>
 +
      <h4><b> DNA/Plasmids</b> </h4><br>
 +
      <ol>
 +
        <li>Negative Control: BBa_R0040</li>
 +
        <li>Positive Control: BBa_I20270</li>
 +
        <li>Test Device 1: BBa_J364000</li>
 +
        <li>Test Device 2: BBa_J364001</li>
 +
        <li>Test Device 3: BBa_J364002</li>
 +
        <li>Test Device 4: BBa_J364007</li>
 +
        <li>Test Device 5: BBa_J364008</li>
 +
        <li>Test Device 6: BBa_J364009</li>
 +
      </ol>
 +
      <h4><b> Apparatus</b> </h4><br>
 +
      <ul>
 +
        <li>96 well plates, black with clear flat bottom (provided by Bluepha Lab)</li>
 +
        <li>Plate reader</li>
 +
        <li>Flow Cytometry</li>
 +
        <li>Incubator at 37˚C</li>
 +
        <li>Ice bucket with ice</li>
 +
        <li>Foil covered 50 ml tube</li>
 +
        <li>Eppendorf tubes</li>
 +
        <li>Micropipettes</li>
 +
        <li>Micropipette tips</li>
 +
      </ul>
 +
      <h4><b> Materials</b> </h4><br>
 +
      <ul>
 +
        <li>Measurement Kit</li>
 +
        <dd> - LUDOX CL-X</dd>
 +
        <dd> - Silica beads</dd>
 +
        <dd> - Fluorescein</dd>
 +
        <li>1x PBS (Phosphate buffered saline, pH 7.4-7.6)</li>
 +
        <li>ddH2O</li>
 +
        <li>Competent cells (Escherichia coli strain DH5α)</li>
 +
        <li>LB (Luria Bertani) media</li>
 +
        <li>Chloramphenicol</li>
 +
        <li>LB plates</li>
 +
        <li>distilled water</li>
 +
      </ul>
 +
    </div>
 +
    <br><br><br>
 +
    <div class="row" style="padding-left:50px">
 +
      <h2> Protocols</h2><br>
 +
      <h4 style="padding-left:50px"> We had been asked to follow the protocols provided by iGEM HQ. </h4>
 +
      <a style="padding-left:50px" href="https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf"> 2018 InterLab Plate Reader Protocol</a> <br>
 +
      <a style="padding-left:50px" href="https://static.igem.org/mediawiki/2018/e/ec/2018_InterLab_Flow_Cytometry_Protocol.pdf"> 2018 InterLab Flow Cytometry Protocol</a> <br>
 +
      <a style="padding-left:50px" href="http://parts.igem.org/Help:Protocols/Transformation"> Help: Protocols/Transformation</a> <br>
 +
 
 +
    </div>
 +
    <br><br><br>
 +
    <div class="row" style="padding-left:50px">
 +
      <h2> Members</h2><br>
 +
      <div class="members" >
 +
      <a href="https://2018.igem.org/Team:GreatBay_China/Team">
 +
      <img class="photo" id='wky' width="250px" src="https://static.igem.org/mediawiki/2018/d/d3/T--GreatBay_China--cs_p.jpg" />
 +
            <h4>Charles Wei</h4>
 +
      </a>
 +
      </div>
 +
      <div class="members" >
 +
      <a href="https://2018.igem.org/Team:GreatBay_China/Team">
 +
      <img class="photo" id='yx' width="250px" src="https://static.igem.org/mediawiki/2018/0/07/T--GreatBay_China--yx.jpg"/>
 +
            <h4>Sean Yao</h4>
 +
      </a>
 +
      </div>
 +
      <div class="members" >
 +
      <a href="https://2018.igem.org/Team:GreatBay_China/Team">
 +
      <img class="photo" id='ych' width="250px" src="https://static.igem.org/mediawiki/2018/1/1e/T--GreatBay_China--ych_p.jpg" />
 +
            <h4>David Yang</h4>
 +
      </a>
 +
      </div>
 +
    </div>
 +
    <br><br><br>
 +
    <div class="row" style="padding-left:50px">
 +
      <h2> Field Work</h2><br>
 +
      <h4 style="padding-left:50px;padding-right:50px;"> For the reason that our plate reader could only measure the absorbance, we collaborated with SZU-China and borrowed their plate reader.</h4> </br>
 +
 
 +
      <h4><b> Day 1</b> </h4><br>
 +
      <ul>
 +
        <li>The plasmids from distribution kit had been resuspended and transformed into Escherichia coli DH5α</li>
 +
      </ul>
 +
 
 +
      <h4><b> Day 2</b> </h4><br>
 +
      <ul>
 +
        <li>Single colonies had been picked from transformation plates and inoculated into LB medium</li>
 +
        <li>All the supplies and reagents for Calibration and Cell measurement had been prepared</li>
 +
      </ul>
 +
      <br>
 +
      <img class="photo" src="https://static.igem.org/mediawiki/2018/8/86/T--GreatBay_China--interlab_f1.jpg" width="700px" style="padding-left: 150px;">
 +
 
 +
      <h4><b> Day 3</b> </h4><br>
 +
      <ul>
 +
        <li>The three calibration had been done during the dilution of overnight cultures</li>
 +
        <li>The Cell measurement had been done following the protocol</li>
 +
        <li>The flow cytometer collected the samples after measured each plate</li>
 +
        <li>Protocol: Colony Forming Units per 0.1 OD600 E. coli cultures had been done</li>
 +
      </ul>
 +
      <br>
 +
      <div class="contact">
 +
      <img src="https://static.igem.org/mediawiki/2018/9/90/T--GreatBay_China--interlab_f2.png" width="400px" style="padding-left: 50px;">
 +
      </div>
 +
      <div class="contact">
 +
      <img src="https://static.igem.org/mediawiki/2018/4/48/T--GreatBay_China--interlab_f3.png" width="400px" style="padding-left: 50px;">
 +
      </div>
 +
 
 +
      <h4><b> Day 4</b> </h4><br>
 +
      <ul>
 +
        <li>Count the colonies (thanks the ColonyCount from promega)</li>
 +
      </ul>
 +
      <br>
 +
      <div class="contact">
 +
      <img src="https://static.igem.org/mediawiki/2018/8/8d/T--GreatBay_China--interlab_f4.png" width="400px" style="padding-left: 100px;">
 +
      </div>
 +
      <div class="contact">
 +
      <img src="https://static.igem.org/mediawiki/2018/8/85/T--GreatBay_China--interlab_f5.png" width="300px" style="padding-left: 75px;">
 +
      </div>
 +
    </div>
 +
    <br><br><br>
 +
    <div class="row" style="padding-left:50px">
 +
      <h2> Results</h2><br>
 +
      <h3><b> Calibrations</b> </h3><br>
 +
      <h4> OD600 reference point:</h4>
 +
      <img src="https://static.igem.org/mediawiki/2018/a/ac/T--GreatBay_China--interlab_f6.png" width="600px" style="padding-left: 200px;"><br><br>
 +
      <h4> Particle Standard Curve:</h4>
 +
      <img src="https://static.igem.org/mediawiki/2018/f/ff/T--GreatBay_China--interlab_f7.png" width="700px" style="padding-left: 150px;"><br><br>
 +
      <h4> Fluorescein Standard Curve:</h4>
 +
      <img src="https://static.igem.org/mediawiki/2018/e/e7/T--GreatBay_China--interlab_f8.png" width="700px" style="padding-left: 150px;"><br><br><br>
 +
 
 +
      <h4><b> Raw Plate Reader Measurements</b> </h4><br>
 +
      <h4> Fluorescence Raw</h4>
 +
      <img src="https://static.igem.org/mediawiki/2018/d/d5/T--GreatBay_China--interlab_f9.png" width="800px" style="padding-left:50px;"><br><br>
 +
      <h4> Abs600 Raw</h4>
 +
      <img src="https://static.igem.org/mediawiki/2018/2/23/T--GreatBay_China--interlab_f10.png" width="800px" style="padding-left:50px;"><br><br><br>
 +
      <br><br><br><br>
 +
 
 +
      <h4> Our data was accepted on 30th July. Congratulations!</h4>
 +
      <img src="https://static.igem.org/mediawiki/2018/b/b2/T--GreatBay_China--interlab_f12.jpeg" width="800px" style="padding-left:50px;"><br>
 +
    </div>
 +
 
 +
    <div class="row" style="padding-left:50px">
 +
      <h2> Acknowledgement</h2><br>
 +
      <img src="https://static.igem.org/mediawiki/2018/a/a9/T--GreatBay_China--interlab_f11.png" width="800px" style="padding-left:50px;"><br>
 +
 
 +
 
 +
 
 +
 
 +
   
 +
     
 +
 
 +
 
 +
 +
 
 +
 
 +
 
 +
      <!--DON'T FORGET TEMPLATE STARTS HERE BY CHARLES WEI (I wonder why I am writing this cuz only Lucinda and I will edit here.)-->
 
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 +
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Latest revision as of 02:25, 18 October 2018

GBC-Interlab


mCATNIP2018@outlook.com
@Igem2018C

Introduction of Interlab

Reliable and repeatable measurement plays an important role as an engineering discipline in synthetic biology. The InterLab Study aims to identify and correct the sources of systematic variability in synthetic biology measurement, thus to improve the measurement tools and methods which can enable labs to reliably build upon others’ work. It will promote the collaboration within the synthetic biology community. Hence, it may propel the synthetic biology to achieve its full potential.

The previous InterLab studies showed that measuring GFP expression in absolute fluorescence units calibrated against a known concentration of fluorescent molecule can greatly reduce the variability in different labs’ measurements.

The Fifth Year InterLab’s goal is to remove another source of variability, the number of cells in the sample, by using new cell-counting method.


GreatBay_China investigation had helped in answering the question:Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?




Supplies and Reagents


DNA/Plasmids


  1. Negative Control: BBa_R0040
  2. Positive Control: BBa_I20270
  3. Test Device 1: BBa_J364000
  4. Test Device 2: BBa_J364001
  5. Test Device 3: BBa_J364002
  6. Test Device 4: BBa_J364007
  7. Test Device 5: BBa_J364008
  8. Test Device 6: BBa_J364009

Apparatus


  • 96 well plates, black with clear flat bottom (provided by Bluepha Lab)
  • Plate reader
  • Flow Cytometry
  • Incubator at 37˚C
  • Ice bucket with ice
  • Foil covered 50 ml tube
  • Eppendorf tubes
  • Micropipettes
  • Micropipette tips

Materials


  • Measurement Kit
    • - LUDOX CL-X
    - Silica beads
    - Fluorescein
  • 1x PBS (Phosphate buffered saline, pH 7.4-7.6)
  • ddH2O
  • Competent cells (Escherichia coli strain DH5α)
  • LB (Luria Bertani) media
  • Chloramphenicol
  • LB plates
  • distilled water



Protocols


We had been asked to follow the protocols provided by iGEM HQ.

2018 InterLab Plate Reader Protocol
2018 InterLab Flow Cytometry Protocol
Help: Protocols/Transformation



Members


Charles Wei

Sean Yao

David Yang



Field Work


For the reason that our plate reader could only measure the absorbance, we collaborated with SZU-China and borrowed their plate reader.


Day 1


  • The plasmids from distribution kit had been resuspended and transformed into Escherichia coli DH5α

Day 2


  • Single colonies had been picked from transformation plates and inoculated into LB medium
  • All the supplies and reagents for Calibration and Cell measurement had been prepared

Day 3


  • The three calibration had been done during the dilution of overnight cultures
  • The Cell measurement had been done following the protocol
  • The flow cytometer collected the samples after measured each plate
  • Protocol: Colony Forming Units per 0.1 OD600 E. coli cultures had been done

Day 4


  • Count the colonies (thanks the ColonyCount from promega)




Results


Calibrations


OD600 reference point:



Particle Standard Curve:



Fluorescein Standard Curve:




Raw Plate Reader Measurements


Fluorescence Raw



Abs600 Raw








Our data was accepted on 30th July. Congratulations!


Acknowledgement