Difference between revisions of "Team:Stuttgart/InterLab"

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<h1 align=middle>Interlab Study</h1>
 
<h1 align=middle>Interlab Study</h1>
 
<div class="container">
 
<div class="container">
−
  <h3>Introduction</h3>
 
 
       <div class="row section">
 
       <div class="row section">
 
         <div class="col-xs-12 col-sm-8 col-md-8">
 
         <div class="col-xs-12 col-sm-8 col-md-8">
 +
          <h2>Introduction</h2>
 +
<br>
 
             <p>Reproducibility of an experiment and reliable measurement results are important parts of synthetic biology but it is very complicated to find a significant number of laboratories that are able to work on a identical protocol to produce comparable results.  
 
             <p>Reproducibility of an experiment and reliable measurement results are important parts of synthetic biology but it is very complicated to find a significant number of laboratories that are able to work on a identical protocol to produce comparable results.  
 
               Being in many places in the world iGEM seems to be the perfect platform to compare results of one experiment in many different laboratories.
 
               Being in many places in the world iGEM seems to be the perfect platform to compare results of one experiment in many different laboratories.
 
iGEM Stuttgart also took part at the fourth InterLab Measurement Study in 2017.  
 
iGEM Stuttgart also took part at the fourth InterLab Measurement Study in 2017.  
 
The aim of this years study was to measure GFP fluorescence of 6 different test devices (+negative and positive control).  
 
The aim of this years study was to measure GFP fluorescence of 6 different test devices (+negative and positive control).  
−
The following eight constructs (iGEM-kit plate 7) were transformed into chemo-competent DH5α E.coli cells:</p>
+
The following eight constructs on the right side (iGEM-kit plate 7) were transformed into chemo-competent DH5α E.coli cells.
 +
First step of the experiment is the calibration of the available plate reader with LUDOX SH40 and the generation of a standard curve with fluorescein (both provided in the iGEM-Measurement Kit).
 +
The next step is the transformation of the mentioned plasmids into DH5alpha E.coli cells followed by the procedure explained in the picture below.
 +
Results of the measurements of OD600 and fluorescence after 0, 2, 4 and 6 hours will be compared and used to establish a precise GFP measurement protocol.
 +
We used a BioTek Synergy 2 plate reader and clear 96-well plates for both measurements.
 +
You can find the protocol, the detailed description of the experiment and information about the InterLab Study itself <a href="https://2017.igem.org/Competition/InterLab_Study">here</a>.
 +
</p>
 
</div>
 
</div>
 
<div class="col-xs-12 col-sm-4 col-md-4">
 
<div class="col-xs-12 col-sm-4 col-md-4">
 +
<br>
 +
<br>
 +
<br>
 
<table>  
 
<table>  
 
   <tr>  
 
   <tr>  
−
         <td>Positive Control (BBa_120270)</td>
+
         <td>Positive Control (BBa_120270)</td></tr>
−
        <td>Negative Control (BBa_R0040)</td>
+
      <tr> <td>Negative Control (BBa_R0040)</td></tr>
−
        <td>Test Device 1 (BBa_J364000)</td>
+
      <tr><td>Test Device 1 (BBa_J364000)</td></tr>
−
        <td>Test Device 2 (BBa_J364001)</td>
+
      <tr> <td>Test Device 2 (BBa_J364001)</td></tr>
−
        <td>Test Device 3 (BBa_J364002)</td>
+
      <tr><td>Test Device 3 (BBa_J364002)</td></tr>
−
         <td>Test Device 4 (BBa_J364003)</td>
+
         <tr><td>Test Device 4 (BBa_J364003)</td></tr>
−
        <td>Test Device 5 (BBa_J364004)</td>
+
      <tr> <td> Test Device 5 (BBa_J364004)</td></tr>
−
        <td>Test Device 6 (BBa_J364005)</td>
+
      <tr> <td>Test Device 6 (BBa_J364005)</td></tr>
−
</tr>
+
 
</table>
 
</table>
 
</div>
 
</div>
 
</div>
 
</div>
 
<div class="row section">
 
<div class="row section">
−
  <div class="col-xs-12 col-sm-7 col-md-7">
+
    <div class="col-xs-12 col-sm-12 col-md-12">
−
<p>First step of the experiment is the calibration of the available plate reader with LUDOX SH40 and the generation of a standard curve with fluorescein (both provided in the iGEM-Measurement Kit).
+
         <!--<img src="https://static.igem.org/mediawiki/2017/6/6b/TeamStuttgart_workflow_platereader2">-->
−
The next step is the transformation of the mentioned plasmids into DH5alpha E.coli cells followed by the procedure explained in the picture below.
+
         <img src="https://static.igem.org/mediawiki/2017/6/6b/TeamStuttgart_workflow_platereader2.png" class="img-responsive"/>
−
Results of the measurements of OD600 and fluorescence after 0, 2, 4 and 6 hours will be compared and used to establish a precise GFP measurement protocol.
+
−
We used a BioTek Synergy 2 plate reader and clear 96-well plates for both measurements.
+
−
You can find the protocol, the detailed description of the experiment and information about the InterLab Study itself <a href="https://2017.igem.org/Competition/InterLab_Study">here</a>.
+
−
</p>
+
−
    </div>
+
−
    <div class="col-xs-12 col-sm-5 col-md-5">
+
−
         <!--<img src="https://static.igem.org/mediawiki/2017/7/77/TeamStuttgart_plasmids.png">-->
+
−
         <img src="https://static.igem.org/mediawiki/2017/7/77/TeamStuttgart_plasmids.png" class="img-responsive"/>
+
 
     </div>
 
     </div>
 
   </div>
 
   </div>
 
</div>
 
</div>
−
 
+
<br>
 +
<br>
 
<div class="container">
 
<div class="container">
−
  <h3>Results and Discussion</h3>
 
 
       <div class="row section">
 
       <div class="row section">
−
        <h4>LUDOX-OD600 Measurement (calibration)</h4>
+
         <div class="col-xs-12 col-sm-7 col-md-7">
−
         <div class="col-xs-12 col-sm-8 col-md-8">
+
          <h2>Results and Discussion</h2>
 +
<br>
 +
<br>
 +
          <h3>LUDOX-OD600 Measurement (calibration)</h3>
 +
<br>
 
             <p>HIER TEXT REINKOPIEREN</p>
 
             <p>HIER TEXT REINKOPIEREN</p>
 
</div>
 
</div>
−
<div class="col-xs-12 col-sm-4 col-md-4">
+
<div class="col-xs-12 col-sm-5 col-md-5">
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 
   <table>  
 
   <table>  
 
     <tr> <th></th> <th>LUDOX-HS40</th><th>H2O</th> </tr>
 
     <tr> <th></th> <th>LUDOX-HS40</th><th>H2O</th> </tr>
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     <tr><td>Replicate 3</td><td>0,043</td><td>0,026</td></tr>
 
     <tr><td>Replicate 3</td><td>0,043</td><td>0,026</td></tr>
 
     <tr><td>Replicate 4</td><td>0,04</td><td>0,028</td></tr>
 
     <tr><td>Replicate 4</td><td>0,04</td><td>0,028</td></tr>
−
  </table>
 
−
  <table>
 
 
     <tr><th>Arith. Mean</th> <th>0,041</th>
 
     <tr><th>Arith. Mean</th> <th>0,041</th>
 
     <tr><td>Corrected Abs600</td><td>0,0143</td></tr>
 
     <tr><td>Corrected Abs600</td><td>0,0143</td></tr>
Line 665: Line 674:
 
</div>
 
</div>
 
<div class="row section">
 
<div class="row section">
−
  <h3>Fluorescein-Fluorescence-Measurement (standard curve)</h3>
+
   <div class="col-xs-12 col-sm-4 col-md-4">
−
   <div class="col-xs-12 col-sm-7 col-md-7">
+
    <h3>Fluorescein-Fluorescence-Measurement (standard curve)</h3>
 +
<br>
 
<p>Measurement of different fluorescein concentrations.
 
<p>Measurement of different fluorescein concentrations.
 
excitation wavelength: 485nm; emission wavelength: 528nm</p>
 
excitation wavelength: 485nm; emission wavelength: 528nm</p>
 
     </div>
 
     </div>
−
     <div class="col-xs-12 col-sm-5 col-md-5">
+
     <div class="col-xs-12 col-sm-8 col-md-8">
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 
         <!--<img src="https://static.igem.org/mediawiki/2017/4/4b/TeamStuttgart_fluorescin.png">-->
 
         <!--<img src="https://static.igem.org/mediawiki/2017/4/4b/TeamStuttgart_fluorescin.png">-->
 
         <img src="https://static.igem.org/mediawiki/2017/4/4b/TeamStuttgart_fluorescin.png" class="img-responsive"/>
 
         <img src="https://static.igem.org/mediawiki/2017/4/4b/TeamStuttgart_fluorescin.png" class="img-responsive"/>
 
     </div>
 
     </div>
 
   </div>
 
   </div>
 +
<br>
 
   <div class="row section">
 
   <div class="row section">
−
    <h3>Transformtaion</h3>
 
 
     <div class="col-xs-12 col-sm-4 col-md-4">
 
     <div class="col-xs-12 col-sm-4 col-md-4">
 +
      <h3>Transformation</h3>
 +
<br>
 
   <p>We had at least two colonies of every test device except for test device 1 (transformation not successful).
 
   <p>We had at least two colonies of every test device except for test device 1 (transformation not successful).
 
All of the following measurements are without test device 1. Agar-plates with colonies after transformation.</p>
 
All of the following measurements are without test device 1. Agar-plates with colonies after transformation.</p>
 
       </div>
 
       </div>
 
       <div class="col-xs-12 col-sm-8 col-md-8">
 
       <div class="col-xs-12 col-sm-8 col-md-8">
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 
           <!--<img src="https://static.igem.org/mediawiki/2017/3/3b/TeamStuttgart_agarplates.jpeg">-->
 
           <!--<img src="https://static.igem.org/mediawiki/2017/3/3b/TeamStuttgart_agarplates.jpeg">-->
 
           <img src="https://static.igem.org/mediawiki/2017/3/3b/TeamStuttgart_agarplates.jpeg" class="img-responsive"/>
 
           <img src="https://static.igem.org/mediawiki/2017/3/3b/TeamStuttgart_agarplates.jpeg" class="img-responsive"/>
 
       </div>
 
       </div>
 
     </div>
 
     </div>
 +
<br>
 
     <div class="row section">
 
     <div class="row section">
−
      <h3>Cell Measurement</h3>
+
       <div class="col-xs-12 col-sm-6 col-md-6">
−
       <div class="col-xs-12 col-sm-7 col-md-7">
+
        <h3>Cell Measurement</h3>
 +
<br>
 
     <p>OD600-measurement - growth of E.coli cells</p>
 
     <p>OD600-measurement - growth of E.coli cells</p>
 
         </div>
 
         </div>
−
         <div class="col-xs-12 col-sm-5 col-md-5">
+
         <div class="col-xs-12 col-sm-6 col-md-6">
 +
<br>
 +
<br>
 +
<br>
 
             <!--<img src="https://static.igem.org/mediawiki/2017/1/1c/TeamStuttgart_bactgrowth.png">-->
 
             <!--<img src="https://static.igem.org/mediawiki/2017/1/1c/TeamStuttgart_bactgrowth.png">-->
 
             <img src="https://static.igem.org/mediawiki/2017/1/1c/TeamStuttgart_bactgrowth.png" class="img-responsive"/>
 
             <img src="https://static.igem.org/mediawiki/2017/1/1c/TeamStuttgart_bactgrowth.png" class="img-responsive"/>
 
         </div>
 
         </div>
 
       </div>
 
       </div>
 +
<br>
 
       <div class="row section">
 
       <div class="row section">
−
        <h3>Fluorescence Measurement of E.coli cells</h3>
+
         <div class="col-xs-12 col-sm-6 col-md-6">
−
         <div class="col-xs-12 col-sm-7 col-md-7">
+
          <h3>Fluorescence Measurement of E.coli cells</h3>
 +
<br>
 
       <p>Excitation wavelength: 485nm; emission wavelength: 528nm</p>
 
       <p>Excitation wavelength: 485nm; emission wavelength: 528nm</p>
 
           </div>
 
           </div>
−
           <div class="col-xs-12 col-sm-5 col-md-5">
+
           <div class="col-xs-12 col-sm-6 col-md-6">
 +
<br>
 +
<br>
 +
<br>
 +
<br>
 
               <!--<img src="https://static.igem.org/mediawiki/2017/f/f2/TeamStuttgart_fluokurven.png">-->
 
               <!--<img src="https://static.igem.org/mediawiki/2017/f/f2/TeamStuttgart_fluokurven.png">-->
 
               <img src="https://static.igem.org/mediawiki/2017/f/f2/TeamStuttgart_fluokurven.png" class="img-responsive"/>
 
               <img src="https://static.igem.org/mediawiki/2017/f/f2/TeamStuttgart_fluokurven.png" class="img-responsive"/>

Revision as of 15:34, 29 October 2017

Interlab Study

Introduction


Reproducibility of an experiment and reliable measurement results are important parts of synthetic biology but it is very complicated to find a significant number of laboratories that are able to work on a identical protocol to produce comparable results. Being in many places in the world iGEM seems to be the perfect platform to compare results of one experiment in many different laboratories. iGEM Stuttgart also took part at the fourth InterLab Measurement Study in 2017. The aim of this years study was to measure GFP fluorescence of 6 different test devices (+negative and positive control). The following eight constructs on the right side (iGEM-kit plate 7) were transformed into chemo-competent DH5α E.coli cells. First step of the experiment is the calibration of the available plate reader with LUDOX SH40 and the generation of a standard curve with fluorescein (both provided in the iGEM-Measurement Kit). The next step is the transformation of the mentioned plasmids into DH5alpha E.coli cells followed by the procedure explained in the picture below. Results of the measurements of OD600 and fluorescence after 0, 2, 4 and 6 hours will be compared and used to establish a precise GFP measurement protocol. We used a BioTek Synergy 2 plate reader and clear 96-well plates for both measurements. You can find the protocol, the detailed description of the experiment and information about the InterLab Study itself here.




Positive Control (BBa_120270)
Negative Control (BBa_R0040)
Test Device 1 (BBa_J364000)
Test Device 2 (BBa_J364001)
Test Device 3 (BBa_J364002)
Test Device 4 (BBa_J364003)
Test Device 5 (BBa_J364004)
Test Device 6 (BBa_J364005)


Results and Discussion



LUDOX-OD600 Measurement (calibration)


HIER TEXT REINKOPIEREN







LUDOX-HS40H2O
Replicate 10,040,026
Replicate 20,0410,027
Replicate 30,0430,026
Replicate 40,040,028
Arith. Mean 0,041
Corrected Abs6000,0143
Reference OD6000,043
OD600/Abs6002,98

Fluorescein-Fluorescence-Measurement (standard curve)


Measurement of different fluorescein concentrations. excitation wavelength: 485nm; emission wavelength: 528nm






Transformation


We had at least two colonies of every test device except for test device 1 (transformation not successful). All of the following measurements are without test device 1. Agar-plates with colonies after transformation.






Cell Measurement


OD600-measurement - growth of E.coli cells





Fluorescence Measurement of E.coli cells


Excitation wavelength: 485nm; emission wavelength: 528nm