Difference between revisions of "Team:Stuttgart/InterLab"

 
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<h1 align=middle>Interlab Study</h1>
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<h1 align=middle>InterLab Study</h1>
 
<div class="container">
 
<div class="container">
 
       <div class="row section">
 
       <div class="row section">
 
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           <h2>Introduction</h2>
 
           <h2>Introduction</h2>
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<br>
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        </div>
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      </div>
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      <div class="row section">
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        <div class="col-xs-12 col-sm-12 col-md-12">
 
             <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.
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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.  
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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).  
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The following eight constructs (iGEM-kit plate 7) were transformed into chemo-competent DH5α E.coli cells:
+
              The eight constructs (iGEM-kit plate 7) listed below were transformed into chemo-competent DH5α <i>E. coli</i> cells.</p>
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<ul>
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          </div>
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  <li>Positive Control (BBa_120270)</li>
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        </div>
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      <li>Negative Control (BBa_R0040)</li>
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    <div class="row section">   
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       <li>Test Device 1 (BBa_J364000)</li>
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<div class="col-xs-12 col-sm-5 col-md-5">
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       <li>Test Device 2 (BBa_J364001)</li>
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    <ul>
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      <li>Test Device 3 (BBa_J364002)</li>
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      <li>Positive Control (<a href="http://parts.igem.org/Part:BBa_120270">BBa_120270</a>)</li>
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        <li>Test Device 4 (BBa_J364003)</li>
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      <li>Negative Control (<a href="http://parts.igem.org/Part:BBa_R0040">BBa_R0040</a>)</li>
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    <li> Test Device 5 (BBa_J364004)</li>
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       <li>Test Device 1 (<a href="http://parts.igem.org/Part:BBa_J364000">BBa_J364000</a>)</li>
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       <li>Test Device 6 (BBa_J364005)</li></ul>
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       <li>Test Device 2 (<a href="http://parts.igem.org/Part:BBa_J364001">BBa_J364001</a>)</li>
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<br>
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      <li>Test Device 3 (<a href="http://parts.igem.org/Part:BBa_J364002">BBa_J364002</a>)</li>
 +
      <li>Test Device 4 (<a href="http://parts.igem.org/Part:BBa_J364003">BBa_J364003</a>)</li>
 +
      <li> Test Device 5 (<a href="http://parts.igem.org/Part:BBa_J364004">BBa_J364004</a>)</li>
 +
       <li>Test Device 6 (<a href="http://parts.igem.org/Part:BBa_J364005">BBa_J364005</a>)</li>
 +
  </ul>
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</div>
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<div class="col-xs-12 col-sm-7 col-md-7">     
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<div class="thumb tleft">
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<div class="thumbinner" style="width:400px;">
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<a href="https://static.igem.org/mediawiki/2017/3/39/Workflow_interlab.png" class="image">
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<img alt="" src="https://static.igem.org/mediawiki/2017/3/39/Workflow_interlab.png" width="380" height="200" class="thumbimage" />
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</a> 
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<div class="thumbcaption">
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<div class="magnify">
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<a href="https://static.igem.org/mediawiki/2017/3/39/Workflow_interlab.png" class="internal" title="Enlarge">
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  <img src="/wiki/skins/common/images/magnify-clip.png" width="15" height="11" alt="" />
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</a>
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</div>
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<b>Figure 1:</b> Workflow InterLab Study, from InterLab_2017_Plate_Reader_Protocol.
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</div>
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</div>
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</div>
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</div>
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</div>
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<div class="row section">
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  <div class="col-xs-12 col-sm-12 col-md-12">
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<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).  
 
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.
+
The next step is the transformation of the mentioned plasmids into DH5alpha <i>E. coli</i> 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.  
 
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.  
 
We used a BioTek Synergy 2 plate reader and clear 96-well plates for both measurements.  
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        <!--<img src="https://static.igem.org/mediawiki/2017/6/6b/TeamStuttgart_workflow_platereader2">-->
 
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<div class="container">  
 
<div class="container">  
 
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        <div class="col-xs-12 col-sm-12 col-md-12">
 
<h2>Results and Discussion</h2>
 
<h2>Results and Discussion</h2>
 
           <h3>LUDOX-OD600 Measurement (calibration)</h3>
 
           <h3>LUDOX-OD600 Measurement (calibration)</h3>
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         <div class="col-xs-12 col-sm-6 col-md-6">
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             <p>HIER TEXT REINKOPIEREN</p>
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             <p>For the calibration 100 μL of LUDOX-S40 from the InterLab Measurement Kit was added into the wells A1, B1, C1, and D1. Analogical 100 μL of ddH<sub>2</sub>O was added to the wells A2, B2, C2, D2.
 +
              The measurement was performed with a plate reader at an absorbance of 600 nm.
 +
</p>
 
</div>
 
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<div class="col-xs-12 col-sm-7 col-md-7">
 
   <table>  
 
   <table>  
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     <tr> <th></th> <th>LUDOX-HS40</th><th>H2O</th> </tr>
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     <tr> <th></th> <th>LUDOX-HS40</th><th>H<sub>2</sub>O</th> </tr>
 
     <tr><td>Replicate 1</td><td>0,04</td><td>0,026</td></tr>
 
     <tr><td>Replicate 1</td><td>0,04</td><td>0,026</td></tr>
 
     <tr><td>Replicate 2</td><td>0,041</td><td>0,027</td></tr>
 
     <tr><td>Replicate 2</td><td>0,041</td><td>0,027</td></tr>
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<div class="row section">
 
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<h3>Fluorescein-Fluorescence-Measurement (standard curve)</h3>
 
<h3>Fluorescein-Fluorescence-Measurement (standard curve)</h3>
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<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>
 
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         <!--<img src="https://static.igem.org/mediawiki/2017/4/4b/TeamStuttgart_fluorescin.png">-->
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         <!--<img src="https://static.igem.org/mediawiki/2017/6/63/Interlab1.png">-->
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         <img src="https://static.igem.org/mediawiki/2017/4/4b/TeamStuttgart_fluorescin.png" class="img-responsive"/>
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         <img src="https://static.igem.org/mediawiki/2017/8/8d/Interabfluoro4.png" class="img-responsive"/>
 
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<br>
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   <div class="row section">
 
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    <div class="col-xs-12 col-sm-12 col-md-12">
 
<h3>Transformation</h3>
 
<h3>Transformation</h3>
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    <div class="col-xs-12 col-sm-4 col-md-4">
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   <p> We had at least two colonies of every test device except for test device 1 (transformation not successful).
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<br>
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All of the following measurements are without test device 1. </p>
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   <p>We had at least two colonies of every test device except for test device 1 (transformation not successful).
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All of the following measurements are without test device 1. Agar-plates with colonies after transformation.</p>
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           <!--<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"/>
 
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<br>
 
 
     <div class="row section">
 
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<h3>Cell Measurement</h3>
 
 
       <div class="col-xs-12 col-sm-6 col-md-6">
 
       <div class="col-xs-12 col-sm-6 col-md-6">
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<br>
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<h3>Cell Measurement</h3>
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     <p>OD600-measurement - growth of E.coli cells</p>
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     <p>OD600-measurement - growth of <i>E. coli cells</i>.<br>All cells (with different parts) show the expectable exponential growth.</p>
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        <div class="col-xs-12 col-sm-6 col-md-6">
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             <!--<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">-->
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             <img src="https://static.igem.org/mediawiki/2017/1/1c/TeamStuttgart_bactgrowth.png" class="img-responsive"/>
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             <img src="https://static.igem.org/mediawiki/2017/6/64/Interabfluoro5.png" class="img-responsive"/>
 
         </div>
 
         </div>
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<br>
 
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      <div class="row section">
 
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<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-6 col-md-6">
−
<br>
+
<h3>Fluorescence Measurement of <i>E. coli</i> cells</h3>
−
       <p>Excitation wavelength: 485nm; emission wavelength: 528nm</p>
+
       <p>Test device 2, the positive control and test device 4 show the highest fluorescence after 6 hours followed by test device 5. The remaining test devices do not show any increasing fluorescence and stay on the level of the negative control.
−
          </div>
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        (Excitation wavelength: 485nm; emission wavelength: 528nm)</p>
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          <div class="col-xs-12 col-sm-6 col-md-6">
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               <!--<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">-->
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               <img src="https://static.igem.org/mediawiki/2017/f/f2/TeamStuttgart_fluokurven.png" class="img-responsive"/>
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               <img src="https://static.igem.org/mediawiki/2017/2/27/Interabfluoro6.png" class="img-responsive"/>
 
           </div>
 
           </div>
 
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Latest revision as of 22:30, 14 December 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 eight constructs (iGEM-kit plate 7) listed below were transformed into chemo-competent DH5α E. coli cells.

Figure 1: Workflow InterLab Study, from InterLab_2017_Plate_Reader_Protocol.

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.

Results and Discussion

LUDOX-OD600 Measurement (calibration)

For the calibration 100 μL of LUDOX-S40 from the InterLab Measurement Kit was added into the wells A1, B1, C1, and D1. Analogical 100 μL of ddH2O was added to the wells A2, B2, C2, D2. The measurement was performed with a plate reader at an absorbance of 600 nm.

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.

Cell Measurement

OD600-measurement - growth of E. coli cells.
All cells (with different parts) show the expectable exponential growth.

Fluorescence Measurement of E. coli cells

Test device 2, the positive control and test device 4 show the highest fluorescence after 6 hours followed by test device 5. The remaining test devices do not show any increasing fluorescence and stay on the level of the negative control. (Excitation wavelength: 485nm; emission wavelength: 528nm)