Difference between revisions of "Team:UCLouvain/InterLab"

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{{UCLouvain}}
 
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<html>
 
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<title>iGEM UCLouvain Team</title>
 
 
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/*
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<div class="site-hero_2">
Our styles
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<div class="page-title">
*/
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<div class="big-title montserrat-text uppercase">In the lab</div>
 +
<div class="small-title montserrat-text uppercase">Inter Lab Study</div>
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</div>
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</div>
  
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<div class="col-md-12">
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<div class="section-title" style="text-align:left;float:left;width:100%;margin-bottom:0">
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<span>TITLE</span>
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<div class="small-title montserrat-text uppercase">SUBTITLE</div>
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<p>PARAGRAPH</p>
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</div>
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</section>
  
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<div class="big-title montserrat-text uppercase">Our Project</div>
 +
<div class="small-title montserrat-text uppercase">Interlab measurement study</div>
 +
</div>
 +
</div>
  
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 +
<span>Introduction</span>
 +
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.igem_2017_content_wrapper h2.smallclear {
+
<p>This being our university’s second participation in the iGEM competition, we’ve now decided to take part in the fourth international InterLaboratory Measurement Study in synthetic biology.</p>
margin-bottom: 5px !important;
+
}
+
<p>This is optional and voluntary, not directly related to our main project (BactaSun) but nonetheless a significant component of the iGEM Bronze Medal criteria.</p>
 +
 +
<p>One major challenge in the field of engineering biology is to standardize measurements in order to compare and share data from different labs across the world using different protocols, reagents, devices, device settings …</p>
 +
 +
<p>The Interlab study is a project run by  iGEM’s measurement committee to develop a reliable and repeatable measurement procedure for the green fluorescent protein (GFP) which is one of the most used marker in synthetic biology.</p>
 +
 +
<p>Fluorescence data are usually difficult to compare since they are reported in different units or are processed differently according to various research groups. The project’s objective is therefore to improve current measurement tools by converting data into absolute units.</p>
 +
</div>
 +
</div>
 +
<div class="row" style="margin-bottom: 40px;">
 +
<div class="col-md-12">
 +
<div class="section-title" style="text-align:left;float:left;width:100%;margin-bottom:0">
 +
<span>Materials</span>
 +
</div>
  
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<li style="color:#999999;margin-left:20px;">2017 InterLab parts</li>
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<li style="color:#999999;margin-left:40px;">Positive control (BBa_I20270)</li>
}
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<li style="color:#999999;margin-left:40px;">Negative control (BBa_R0040)</li>
 +
<li style="color:#999999;margin-left:40px;">Test Device 1 (BBa_J364000)</li>
 +
<li style="color:#999999;margin-left:40px;">Test Device 2 (BBa_J364001)</li>
 +
<li style="color:#999999;margin-left:40px;">Test Device 3 (BBa_J364002)</li>
 +
<li style="color:#999999;margin-left:40px;">Test Device 4 (BBa_J364003)</li>
 +
<li style="color:#999999;margin-left:40px;">Test Device 5 (BBa_J364004)</li>
 +
<li style="color:#999999;margin-left:40px;">Test Device 6 (BBa_J364005)</li>
 +
</ul>
 +
<li style="color:#999999;margin-left:20px;">Competent Escherichia coli K-12 DH5-alpha cell</li>
 +
<li style="color:#999999;margin-left:20px;">Plate reader: Tecan Infinite® 200 PRO</li>
 +
<li style="color:#999999;margin-left:20px;">96 - wells plate</li>
 +
<li style="color:#999999;margin-left:20px;">InterLab Measurement Kit</li>
 +
</ul>
 +
</div>
 +
</div>
 +
<div class="row" style="margin-bottom: 40px;">
 +
<div class="col-md-12">
 +
<div class="section-title" style="text-align:left;float:left;width:100%;margin-bottom:0">
 +
<span>Methods</span>
 +
</div>
  
.igem_2017_content_wrapper ul {
+
<p>First of all the device needs calibrating before measuring fluorescence. The goal is to determine the optimal settings for the plate reader to detect the optical density at 600nm (OD600 reference point). In order to do so we established a correction factor using LUDOX.</p>
margin: 0 !important;
+
padding: 0 !important;
+
<p>Then we proceed to establish a fluorescein fluorescence standard curve using 4 replicates of fluorescein dilution series. This will allow us to convert cell based readings to an equivalent fluorescein concentration and finally into a GFP concentration.</p>
}
+
 +
<p>As to the cell culture measurements, controls and devices were transformed into DH5-alpha E.coli strain. Two colonies were picked and grown in LB + chloramphenicol overnight at 37°C. Colonies were diluted to the correct OD600 and (subsequently) samples were then measured every 2 hours for 6 hours.</p>
 +
 +
<p>We used an excitation wavelength of 485nm and an emission wavelength of 530nm to measure the GFP activity.</p>
 +
 +
<p>The whole protocol is available at this link: <a style="color:#999999;" href="https://static.igem.org/mediawiki/2017/8/85/InterLab_2017_Plate_Reader_Protocol.pdf">PDF</a>.</p>
 +
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 +
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 +
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<div class="col-md-12">
 +
<div class="section-title" style="text-align:left;float:left;width:100%;margin-bottom:0">
 +
<span>Results</span>
 +
</div>
 +
 +
<div class="section-title" style="text-align:left;float:left;width:100%;margin-bottom:0">
 +
<p class="montserrat-text uppercase">OD600 reference point</p>
 +
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<p>OD600/Abs600 (correcting factor) = 3.125</p>
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<p class="montserrat-text uppercase">Colony 1 cell measurements</p>
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                <ul>
 
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<a href="#">Our Project</a>
 
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<li><a href="#">Approach 2</a></li>
 
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<p>TEXTE</p>
 
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--><li><a href="https://uclouvain.be/en/index.html" target="_blank"><img src="https://static.igem.org/mediawiki/2017/8/87/Logo-UCL.jpg" alt="UCL"></a></li><!--
 
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--><li><a href="http://www.agilent.com/home" target="_blank"><img src="https://static.igem.org/mediawiki/2017/8/88/Logo-Agilent_Technologies.png" alt="Agilent Technologies"></a></li><!--
 
--><li><a href="https://www.aglouvain.be/site2/" target="_blank"><img src="https://static.igem.org/mediawiki/2017/5/5d/Logo-AGL.png" alt="AGL"></a></li><!--
 
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--><li><a href="https://uclouvain.be/en/faculties/sc" target="_blank"><img src="https://static.igem.org/mediawiki/2017/0/01/Logo-UCL_Sciences.jpg" alt="UCL faculté des Sciences"></a></li><!--
 
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Revision as of 23:26, 30 October 2017

iGEM UCLouvain Team iGEM UCLouvain Team

In the lab
Inter Lab Study
TITLE
SUBTITLE

PARAGRAPH

Our Project
Interlab measurement study
Introduction

This being our university’s second participation in the iGEM competition, we’ve now decided to take part in the fourth international InterLaboratory Measurement Study in synthetic biology.

This is optional and voluntary, not directly related to our main project (BactaSun) but nonetheless a significant component of the iGEM Bronze Medal criteria.

One major challenge in the field of engineering biology is to standardize measurements in order to compare and share data from different labs across the world using different protocols, reagents, devices, device settings …

The Interlab study is a project run by iGEM’s measurement committee to develop a reliable and repeatable measurement procedure for the green fluorescent protein (GFP) which is one of the most used marker in synthetic biology.

Fluorescence data are usually difficult to compare since they are reported in different units or are processed differently according to various research groups. The project’s objective is therefore to improve current measurement tools by converting data into absolute units.

Materials
  • 2017 InterLab parts
    • Positive control (BBa_I20270)
    • 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)
  • Competent Escherichia coli K-12 DH5-alpha cell
  • Plate reader: Tecan Infinite® 200 PRO
  • 96 - wells plate
  • InterLab Measurement Kit
Methods

First of all the device needs calibrating before measuring fluorescence. The goal is to determine the optimal settings for the plate reader to detect the optical density at 600nm (OD600 reference point). In order to do so we established a correction factor using LUDOX.

Then we proceed to establish a fluorescein fluorescence standard curve using 4 replicates of fluorescein dilution series. This will allow us to convert cell based readings to an equivalent fluorescein concentration and finally into a GFP concentration.

As to the cell culture measurements, controls and devices were transformed into DH5-alpha E.coli strain. Two colonies were picked and grown in LB + chloramphenicol overnight at 37°C. Colonies were diluted to the correct OD600 and (subsequently) samples were then measured every 2 hours for 6 hours.

We used an excitation wavelength of 485nm and an emission wavelength of 530nm to measure the GFP activity.

The whole protocol is available at this link: PDF.

Results

OD600 reference point

OD600/Abs600 (correcting factor) = 3.125

Fluorescein fluorescence standard curve

Colony 1 cell measurements

you think we're cool? so are our sponsors!
  • UCL
  • Fédération Wallonie Bruxelles
  • Fondation Louvain
  • Imperial College
  • Agilent Technologies
  • AGL
  • ISV
  • UCL faculté des Sciences