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<h1>Attributions</h1>
 
  
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<p> Each team must clearly attribute work done by the student team members on this page. The team must distinguish work done by the students from work done by others, including the host labs, advisors, instructors, and individuals not on the team roster.  
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<br><br>
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This is a bronze medal requirement. Please see the <a href="https://2017.igem.org/Judging/Medals">Medals requirements page</a> for more details.</p>
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<h5> Why is this page needed? </h5>
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<p>The Attribution requirement helps the judges know what you did yourselves and what you had help with. We don't mind if you get help with difficult or complex techniques, but you must report what work your team did and what work was done by others.</p>
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For example, you might choose to work with an animal model during your project. Working with animals requires getting a license and applying far in advance to conduct certain experiments in many countries. This is difficult to achieve during the course of a summer, but much easier if you can work with a postdoc or PI who has the right licenses.</p>
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<h5> What should this page have?</h5>
 
  
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<ul>
 
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<li>General Support</li>
 
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<li>Project support and advice</li>
 
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<li>Fundraising help and advice</li>
 
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<li>Lab support</li>
 
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<li>Difficult technique support</li>
 
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<li>Project advisor support</li>
 
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<li>Wiki support</li>
 
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<li>Presentation coaching</li>
 
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<li>Human Practices support</li>
 
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<li> Thanks and acknowledgements for all other people involved in helping make a successful iGEM team</li>
 
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</ul>
 
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</div>
 
  
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      <div class="Hauptnavigation">
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          <a href="https://2017.igem.org/Team:Tuebingen/Team">Team</a>
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          <a href="https://2017.igem.org/Team:Tuebingen/Inspiration">Inspiration</a>
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          <a href="https://2017.igem.org/Team:Tuebingen/Result">Result</a>
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          <a href="https://2017.igem.org/Team:Tuebingen/Human Practice">Human Practice</a>
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          <a href="https://2017.igem.org/Team:Tuebingen/Lab">Lab</a>
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          <a href="https://2017.igem.org/Team:Tuebingen/Attribution">Attribution</a>
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      <nav class="Unternavigation-Team">
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              <a href="#Introduction">Introduction </a> <br>
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              <a href="#Theoretical-Background">Theoretical Background </a> <br>
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              <a href="#Practical-Workflow">Practical Workflow </a><br>
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              <a href="#Results-and-Discussion">Results and Discussion </a><br>
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      </nav>
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          <img src="https://static.igem.org/mediawiki/2017/8/8c/T--Tuebingen--LabTitle.jpg" alt="InterLabBild" id="BigImageLab">
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          <!-- Content der Seite -->
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                <h1 id="The-Fourth-Interlab-Study">The fourth InterLab Study</h1>
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                <h2 id="Introduction" class="anchor">INTRODUCTION</h2>       
 +
               
 +
                <p>How similar can fluorescence measurements be if the same protocol is used all over the world? This question will be answered in the fourth International InterLab Measurement Study for iGEM 2017. For researchers it is important to standardize protocols to produce reproducible data. Fluorescence values measured from GFP and other fluorochromes are usually difficult to compare as different devices and methods give different values in different units. This year's InterLab Study focusses on a comparable measurement of fluorescence by establishing a step by step protocol for plate readers used by all iGEM teams.</p>             
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                <h2 id="Theoretical-Background" class="anchor"> THEORETICAL  BACKGROUND </h2>       
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 +
                <p>Teams are provided with the same protocol to measure GFP fluorescence with a plate reader. Eight different devices were tested which are listed below:</p>
 +
                  <a href="http://parts.igem.org/Part:BBa_I20270" id="Positive-Control"> ● Positive Control (BBa_I20270)</a><br>
 +
                  <a href="http://parts.igem.org/Part:BBa_R0040"  id="negative-control"> ● Negative Control (BBa_R0040)</a><br>
 +
                  <a href="http://parts.igem.org/Part:BBa_J364000" id="Test-Device1">  ● Test Device 1 (BBa_J364000): J23101.BCD2.E0040.B0015</a><br>
 +
                  <a href="http://parts.igem.org/Part:BBa_J364001" id="Test-Device2">  ● Test Device 2 (BBa_J364001): J23106.BCD2.E0040.B0015</a><br>
 +
                  <a href="http://parts.igem.org/Part:BBa_J364002" id="Test-Device3">  ● Test Device 3 (BBa_J364002): J23117.BCD2.E0040.B0015</a><br>
 +
                  <a href="http://parts.igem.org/Part:BBa_J364003" id="Test-Device4">  ● Test Device 4 (BBa_J364003): J23101+I13504</a><br>
 +
                  <a href="http://parts.igem.org/Part:BBa_J364004" id="Test-Device5">  ● Test Device 5 (BBa_J364004): J23106+I13504</a><br>
 +
                  <a href="http://parts.igem.org/Part:BBa_J364005" id="Test-Device6">  ● Test Device 6 (BBa_J364005): J23117+I13504</a><br>
 +
                <p><br> All test devices  are composite parts containing GFP with constitutive promoters,  a negative control without GFP is also included. The vector pSB1C3 has a chloramphenicol resistance. Device 4, 5, and 6 additionally contain a Bicistronic Design Element Number 2 which was designed by Mutalik et al. in a2013 Nature publication. This element should induce precise and reliable gene expression.
  
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<div class="clear"></div>
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<br><br> Mutalik, V. K. et al. "Precise and reliable gene expression via standard transcription and translation initiation elements." Nature Methods 10, 354–360 (2013).
  
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<div class="column half_size">
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<br><br>  For normalization standard curves were made with the provided measurement kit from iGEM.
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</p>
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                  <br> <br>
 +
                  <h2 id="Practical-Workflow" class="anchor">PRACTICAL WORKFLOW</h2>
 +
                  <p>Before the actual measurement, calibration was performed for OD600 and a fluorescence standard curve was determined using a clear bottom black 96-well plate in four replicates.</p>
 +
                 
 +
                  <h5>Table 1: Instrument settings for calibration</h5>
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                  <table style="width:100%">
 +
  <tr>
 +
    <th>Instrument Settings</th>
 +
    <th>OD6000 reference point
 +
        LUDOX-S40</th>
 +
    <th>fluorescein fluorescence standard curve</th>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>Positioning delay</td>
 +
    <td>0.5 s</td>
 +
    <td>0.2</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>Number of flashes per well</td>
 +
    <td>20</td>
 +
    <td>25</td>
 +
  </tr>
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 +
  <tr>
 +
    <td>Orbital/pathlength correction</td>
 +
    <td>off</td>
 +
    <td>off</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>Optic</td>
 +
    <td>top</td>
 +
    <td>top</td>
 +
   
 +
    <tr>
 +
    <td>gain</td>
 +
    <td></td>
 +
    <td>700</td>
 +
  </tr>
 +
  <tr>
 +
    <td>Excitation</td>
 +
    <td></td>
 +
    <td>485</td>
 +
  </tr>
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  <tr>
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    <td>Emission</td>
 +
    <td></td>
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    <td>520</td>
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  </tr>
 +
  <tr>
 +
    <td>Orbital/pathlength correction</td>
 +
    <td>off</td>
 +
    <td>off</td>
 +
  </tr>
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</table>
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 +
                  <br><br><br>
 +
               
 +
                 
 +
                  <img src="https://static.igem.org/mediawiki/2017/b/b5/T--Tuebingen--Interlabstudy-Fluorescein.png" id="Fluorescin-Standard-Curve">
 +
                  <h5> Figure 1: Fluorescein standard curve obtained by dilution series of fluorescein in 4 replicates. </h5>
 +
                 
 +
                  <br>
 +
               
 +
                  <p> Subsequently, we performed , the actual measurement of 8 different devices  as shown in figure 2. <br> First, plasmids were transformed in DH5-alpha using the standard transformation protocol from iGEM with the deviation of using LB medium instead of SOC medium. For further information on the used protocol go to "http://parts.igem.org/Help:Protocols/Transformation". <br>
 +
                  <br> Two colonies were picked for each device and incubated in 5-10 mL LB medium + Chloramphenicol (25 µg/mL). The next day the solution was diluted to an OD of 0.02 and 500 µL of the samples were taken and hold on ice at t=0, 2, 4, 6 h. Absorbance (OD600) and fluorescence were then measured using the FLUOstar OPTIMA from BMG LABTECH.
 +
                  <a href="https://static.igem.org/mediawiki/2017/8/85/InterLab_2017_Plate_Reader_Protocol.pdf"> (For detailed protocol click here.)</a></p>
 +
                 
 +
                  <img src="https://static.igem.org/mediawiki/2017/f/fb/T--Tuebingen--Interlabstudy-Workflow.png" id="Workflow-InterLab-Study-2017">
 +
                  <h5> Figure 2: Workflow InterLab Study 2017 </h5>
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 +
               
 +
                  <h2 id="Results-and-Discussion" class="anchor"> RESULTS AND DISCUSSION </h2>
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                  <p>The provided protocol by iGEM was easy to implement by providing a step by step guide to perform the experiments. <br><br>
  
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<div class="highlight">
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                      Although our data has a high variance between the devices and between the replicates after normalization, device 1 and 2 showed significant higher fluorescence than device 3. This is in line with the data from the device’s reference in the Registry where device 1 was shown to have the highest absorption followed by device 2 and then device 3.
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<h5> Can we base our project on a previous one? </h5>
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                  </p>
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<p>Yes! You can have a project based on a previous team, or based on someone else's idea, <b>as long as you state this fact very clearly and give credit for the original project.</b> </p>
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</div>
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                  <h5>Table 2: Variant RFP with corresponding absorption values</h5>
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</div>
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                  <table style="width:100%">
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  <tr>
 +
    <th></th>
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    <th>Variant RFP</th>
 +
    <th>Absorption / AU oder mAU</th>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>Device 1</td>
 +
    <td>J23101</td>
 +
    <td>1791</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>Device 2</td>
 +
    <td>J23106</td>
 +
    <td>1185</td>
 +
  </tr>
 +
 
 +
  <tr>
 +
    <td>Device 3</td>
 +
    <td>J23117</td>
 +
    <td>162</td>
 +
  </tr>
 +
                  </table>
 +
                      <p>Device 4, 5 and 6 with the Bicistronic Design Element Number 2 showed no real difference in comparison to device 1, 2 and 3 where this element was not present. When the data from all teams is compared we will see if there is a bigger influence on gene expression due to the different promoters used.<br>
 +
                      At time point 2 h the fluorescence signal was the highest despite for the positive control. If the expression of RFP induces stress, one explanation might be that the bacteria induce expression of proteases or reduce the amount of the necessary transcription factors.
 +
                      </p>
 +
                 
 +
                  <img src=https://static.igem.org/mediawiki/2017/9/9b/T--Tuebingen--Interlabstudy-Data.png id="Results-Fluorescein">
 +
                  <h5>Figure 3: Results show in µM Fluorescein/OD600 for Devices 1, 2, 3 in comparison  to devices 4, 5, 6. Samples were taken at t = 0, 2, 4, 6 h.  Values smaller than 0 were excluded in the graphic. Biological duplicates are represented from each device. BCD2: Bicistronic Design Element Number 2.</h5>
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<h5>Inspiration</h5>
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<p>Take a look at what other teams have done:</p>
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<ul>
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<li><a href="https://2011.igem.org/Team:Imperial_College_London/Team">2011 Imperial College London</a> (scroll to the bottom)</li>
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<li><a href="https://2014.igem.org/Team:Exeter/Attributions">2014 Exeter </a></li>
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<li><a href="https://2014.igem.org/Team:Melbourne/Attributions">2014 Melbourne </a></li>
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<li><a href="https://2014.igem.org/Team:Valencia_Biocampus/Attributions">2014 Valencia Biocampus</a></li>
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<h5>Team training and Project start</h5>
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<p>Tell us if your institution teaches an iGEM or synthetic biology class and when you started your project:</p>
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<ul>
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<li>Does your institution teach an iGEM or synthetic biology course?</li>
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<li>When did you start this course?</li>
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<li>Are the syllabus and course materials freely available online?</li>
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Revision as of 17:10, 1 November 2017

iGem Tübingen 2017

Team Inspiration Result Human Practice Lab Attribution
InterLabBild

The fourth InterLab Study

INTRODUCTION

How similar can fluorescence measurements be if the same protocol is used all over the world? This question will be answered in the fourth International InterLab Measurement Study for iGEM 2017. For researchers it is important to standardize protocols to produce reproducible data. Fluorescence values measured from GFP and other fluorochromes are usually difficult to compare as different devices and methods give different values in different units. This year's InterLab Study focusses on a comparable measurement of fluorescence by establishing a step by step protocol for plate readers used by all iGEM teams.

THEORETICAL BACKGROUND

Teams are provided with the same protocol to measure GFP fluorescence with a plate reader. Eight different devices were tested which are listed below:

● Positive Control (BBa_I20270)
● Negative Control (BBa_R0040)
● Test Device 1 (BBa_J364000): J23101.BCD2.E0040.B0015
● Test Device 2 (BBa_J364001): J23106.BCD2.E0040.B0015
● Test Device 3 (BBa_J364002): J23117.BCD2.E0040.B0015
● Test Device 4 (BBa_J364003): J23101+I13504
● Test Device 5 (BBa_J364004): J23106+I13504
● Test Device 6 (BBa_J364005): J23117+I13504


All test devices are composite parts containing GFP with constitutive promoters, a negative control without GFP is also included. The vector pSB1C3 has a chloramphenicol resistance. Device 4, 5, and 6 additionally contain a Bicistronic Design Element Number 2 which was designed by Mutalik et al. in a2013 Nature publication. This element should induce precise and reliable gene expression.

Mutalik, V. K. et al. "Precise and reliable gene expression via standard transcription and translation initiation elements." Nature Methods 10, 354–360 (2013).

For normalization standard curves were made with the provided measurement kit from iGEM.



PRACTICAL WORKFLOW

Before the actual measurement, calibration was performed for OD600 and a fluorescence standard curve was determined using a clear bottom black 96-well plate in four replicates.

Table 1: Instrument settings for calibration
Instrument Settings OD6000 reference point LUDOX-S40 fluorescein fluorescence standard curve
Positioning delay 0.5 s 0.2
Number of flashes per well 20 25
Orbital/pathlength correction off off
Optic top top
gain 700
Excitation 485
Emission 520
Orbital/pathlength correction off off



Figure 1: Fluorescein standard curve obtained by dilution series of fluorescein in 4 replicates.

Subsequently, we performed , the actual measurement of 8 different devices as shown in figure 2.
First, plasmids were transformed in DH5-alpha using the standard transformation protocol from iGEM with the deviation of using LB medium instead of SOC medium. For further information on the used protocol go to "http://parts.igem.org/Help:Protocols/Transformation".

Two colonies were picked for each device and incubated in 5-10 mL LB medium + Chloramphenicol (25 µg/mL). The next day the solution was diluted to an OD of 0.02 and 500 µL of the samples were taken and hold on ice at t=0, 2, 4, 6 h. Absorbance (OD600) and fluorescence were then measured using the FLUOstar OPTIMA from BMG LABTECH. (For detailed protocol click here.)

Figure 2: Workflow InterLab Study 2017

RESULTS AND DISCUSSION

The provided protocol by iGEM was easy to implement by providing a step by step guide to perform the experiments.

Although our data has a high variance between the devices and between the replicates after normalization, device 1 and 2 showed significant higher fluorescence than device 3. This is in line with the data from the device’s reference in the Registry where device 1 was shown to have the highest absorption followed by device 2 and then device 3.

Table 2: Variant RFP with corresponding absorption values
Variant RFP Absorption / AU oder mAU
Device 1 J23101 1791
Device 2 J23106 1185
Device 3 J23117 162

Device 4, 5 and 6 with the Bicistronic Design Element Number 2 showed no real difference in comparison to device 1, 2 and 3 where this element was not present. When the data from all teams is compared we will see if there is a bigger influence on gene expression due to the different promoters used.
At time point 2 h the fluorescence signal was the highest despite for the positive control. If the expression of RFP induces stress, one explanation might be that the bacteria induce expression of proteases or reduce the amount of the necessary transcription factors.

Figure 3: Results show in µM Fluorescein/OD600 for Devices 1, 2, 3 in comparison to devices 4, 5, 6. Samples were taken at t = 0, 2, 4, 6 h. Values smaller than 0 were excluded in the graphic. Biological duplicates are represented from each device. BCD2: Bicistronic Design Element Number 2.