Difference between revisions of "Team:Pasteur Paris/InterLab"

 
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<p><a href="#First" class="link">First Approach</a></p>
 
<p><a href="#First" class="link">First Approach</a></p>
 
<p><a href="#Second" class="link">Second Approach</a></p>
 
<p><a href="#Second" class="link">Second Approach</a></p>
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                                <p><a href="#Conclusion" class="link">Conclusion</a></p>
 
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<div class="block title" id="Introduction"><h1>INTRODUCTION</h1></div>
 
<div class="block title" id="Introduction"><h1>INTRODUCTION</h1></div>
 
<div class="block full">
 
<div class="block full">
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<i><p>This year, the iGEM Measurement Committee offered to all teams the possibility to do the Fifth International InterLaboratory Measurement Study in synthetic biology. This work concerns the reliability and repeatability of scientific measurements. </p>
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<i><p>This year, the iGEM Measurement Committee offers to all teams the possibility to do the Fifth International InterLaboratory Measurement Study in synthetic biology. This work concerns the reliability and repeatability of scientific measurements. </p>
 
<p>This project involves <b>providing the same protocols</b> and <b>gathering all data from different teams</b> to build a database with reference values.</p>
 
<p>This project involves <b>providing the same protocols</b> and <b>gathering all data from different teams</b> to build a database with reference values.</p>
−
<p>During this edition, the main objective is to enhance measurements precision in synthetic biology by detecting and correcting sources of errors. Last year, the goal was to reduce variability in fluorescence measurements (GFP) with a normalization of OD. This year, we try to reduce this variability between labs by normalizing to absolute CFUs (colony-forming units). </p></i>
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<p>During this edition, the main objective is to enhance measurements precision in synthetic biology by detecting and correcting sources of errors. Last year, the goal was to reduce variability in fluorescence measurements (GFP) with a normalization of optical density (OD). This year, we tried to reduce this variability between labs by normalizing to absolute colony-forming units (CFUs). <br>
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The iGEM Sorbonne Université team gave us non-competent DH5-<FONT face="Raleway">α</FONT>
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strain. <a href="https://2018.igem.org/Team:Pasteur_Paris/Collaborations#others"style="font-weight: bold ; color:#85196a;"target="_blank">See the collaboration here.</a></p></i>
 
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For this study, we had to transform six plasmids and two controls: <br>
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For this study, we had to transform six plasmids and two controls. We used a protocol of thermocompetence by CaCl2. <br>
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<ul style="text-align: left;">
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<li>Negative control BBa_R0040: sequence for pTet inverting regulator, corresponding to TetR repressible promoter.</li>
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<li><b>Negative control</b> BBa_R0040: sequence for pTet inverting regulator, corresponding to TetR repressible promoter.</li><br>
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<li>Positive control BBa_I20270: promoter and GFP sequence.</li>
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<li><b>Positive control</b> BBa_I20270: promoter and GFP sequence.</li><br>
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<li>And six GFP expressing constitutive devices: <br>Device 1 BBa_J364000 <br> Device 2 BBa_J364001 <br> Device 3 BBa_J364002 <br> Device 4 BBa_J364007 <br> Device 5 BBa_J364008 <br> Device 6 BBa_J364009.</li>
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<li>And six GFP expressing constitutive devices:<br> <br><b>Device 1</b> BBa_J364000 <br><b> Device 2</b> BBa_J364001 <br><b> Device 3</b> BBa_J364002 <br><b> Device 4</b> BBa_J364007 <br><b> Device 5</b> BBa_J364008 <br><b> Device 6</b> BBa_J364009.</li><br>
 
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<div class="block title" id="First">
 
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<h1>FIRST APPROACH<br></h1>
 
<h1>FIRST APPROACH<br></h1>
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<p><i>The first approach consists in converting between absorbance of cells to absorbance of a known concentration of beads.</i></p>
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<p><i>The first approach consists in a conversion between cells absorbance to absorbance of a known concentration of beads.</i></p>
 
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<div class="block title"><h3 style="text-align: left;">Absorbance of a known concentration of beads</h3></div>
 
<div class="block title"><h3 style="text-align: left;">Absorbance of a known concentration of beads</h3></div>
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<td>LUDOX CL-X</td>
 
<td>LUDOX CL-X</td>
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<td>H<sub>2</sub>O</td>
 
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<p>Secondly, we multiplied the colony count by the final dilution factor. Then, we obtained the CFU/mL. </p>
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<p>Then, we multiplied the colony count by the final dilution factor. Doing so, we obtained the CFU/mL. </p>
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<td>Sample</td>
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<td>CFU/mL</td>
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<div class="block title" id="Conclusion"><h1>CONCLUSION</h1></div>
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<td>1.1</td>
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<td>2.72 10<sup>7</sup></td>
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<p>The Interlab experience enabled us to understand the variability of values within machines. Students from Chimie ParisTech performed the Interlab and they discovered new biological methods. Our results were accepted and we hope they will help researchers worldwide to better deal with variability through reading this publication and the results from the other labs. </p>
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Latest revision as of 14:55, 10 November 2018

""

INTRODUCTION

This year, the iGEM Measurement Committee offers to all teams the possibility to do the Fifth International InterLaboratory Measurement Study in synthetic biology. This work concerns the reliability and repeatability of scientific measurements.

This project involves providing the same protocols and gathering all data from different teams to build a database with reference values.

During this edition, the main objective is to enhance measurements precision in synthetic biology by detecting and correcting sources of errors. Last year, the goal was to reduce variability in fluorescence measurements (GFP) with a normalization of optical density (OD). This year, we tried to reduce this variability between labs by normalizing to absolute colony-forming units (CFUs).
The iGEM Sorbonne Université team gave us non-competent DH5-α strain. See the collaboration here.

DEVICES

For this study, we had to transform six plasmids and two controls. We used a protocol of thermocompetence by CaCl2.

  • Negative control BBa_R0040: sequence for pTet inverting regulator, corresponding to TetR repressible promoter.

  • Positive control BBa_I20270: promoter and GFP sequence.

  • And six GFP expressing constitutive devices:

    Device 1 BBa_J364000
    Device 2 BBa_J364001
    Device 3 BBa_J364002
    Device 4 BBa_J364007
    Device 5 BBa_J364008
    Device 6 BBa_J364009.

FIRST APPROACH

The first approach consists in a conversion between cells absorbance to absorbance of a known concentration of beads.

Absorbance of a known concentration of beads

First, we did a calibration, using LUDOX CL-X, to obtain a conversion factor (Figure 1). This factor enables us to transform absorbance data from our plate reader into a basic OD measurement which can be found in a spectrophotometer.

LUDOX CL-X H2O
Replicate 1 0.085 0.069
Replicate 2 0.085 0.066
Replicate 3 0.086 0.064
Replicate 4 0.090 0.064
Arith. Mean 0.087 0.066
Corrected Abs600 0.022 /
Reference OD600 0.063 /
OD600/Abs600 2.930 /
Figure 1: Results of the first calibration. The experiment was performed 4 times. The row colored in red shows our conversion factor

Then, we carried out a second calibration, using silica beads in a microsphere suspension, to convert absorbance measurements into a number of cells. This conversion is based on the plotting of a standard curve of particle concentration (Figure 2) that we have determined during this calibration.

Figure 2: Our particle standard curve based on silica beads measurements.



We did the same work creating a standard curve of fluorescence for fluorescein concentration. Then, we had to use this to transform our cell based readings into a fluorescein concentration.

Figure 3: Our fluorescein standard curve based on fluorescence measurements.

Absorbance of cells

After calibrations, we began cell culture expression measurement. For that, we did overnight cultures of two colonies for each device. 24 hours later, we measured absorbance and fluorescence of growing cultures with our plate reader. Then, we used our fluorescence standard curve to transform our cell measurements into fluorescein concentrations (Figure 4).

Hour 0

Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6
104 165 496 237 -13 444 237 216
35 129 457 252 -50 381 137 150
39 111 504 197 -51 433 209 156
-24 118 426 156 -17 -92 156 170
47 149 612 279 -40 581 240 177
61 122 588 258 -34 564 166 132
61 138 604 281 -44 576 224 156
59 119 559 239 -27 524 154 112


Hour 6

Neg. Control Pos. Control Device 1 Device 2 Device 3 Device 4 Device 5 Device 6
256 2874 8585 3577 175 13888 1562 2137
247 2800 8638 3645 193 14119 1481 2142
265 2874 8618 3729 180 14152 1532 2024
177 2803 8627 3587 72 13714 1526 2057
211 3158 9642 4127 193 12911 1275 1784
234 3272 9661 4206 209 13103 1352 1828
222 3224 9749 4032 203 13199 1354 1862
185 3149 9730 4155 177 12921 1327 1798
Figure 4: Results for fluorescence per particle at hour 0 and hour 6.


According to those results, we can deduce the strength of each promoter.
Device 1 (BBa_J364000): high strength
Device 2 (BBa_J364001): high strength
Device 3 (BBa_J364002): low strength
Device 4 (BBa_J364007): high strength
Device 5 (BBa_J364008): medium strength
Device 6 (BBa_J364009): medium strength

SECOND APPROACH

This second approach involves counting how many colonies grow on a plate. With this number, we have determined a cell concentration for each sample. Then, with CFU values for positive and negative control samples, we calculated a conversion factor from absorbance to CFU.

Example for a final dilution factor of 8.105

First, we counted the colonies on each plate.

Sample Number of colonies
1.1 34
1.2 73
1.3 93
2.1 137
2.2 71
2.3 64
3.1 44
3.2 40
3.3 65

Then, we multiplied the colony count by the final dilution factor. Doing so, we obtained the CFU/mL.

CONCLUSION

The Interlab experience enabled us to understand the variability of values within machines. Students from Chimie ParisTech performed the Interlab and they discovered new biological methods. Our results were accepted and we hope they will help researchers worldwide to better deal with variability through reading this publication and the results from the other labs.