Difference between revisions of "Team:Paris Bettencourt/Contribution"

 
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<h1>Protocol design for PHA production characterisation BBa_K1149051</h1>
 
<h1>Protocol design for PHA production characterisation BBa_K1149051</h1>
  
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<div class=text2><div class=text2left>We decided to characterize the biobrick <a href=”http://parts.igem.org/Part:BBa_K1149051”>BBa_K1149051</a> (<a href=”https://2013.igem.org/Team:Imperial_College”>Imperial College London 2013</a>) using flow cytometry. By staining our cells with a Nile Red solution (0.3mg/mL of DMSO), this technique of Fluorescence-activated cell sorting allowed us to measure the intensity of the Nile Red stained cell containing P3HB. </br></br></div></div>
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<div class=text2><div class=text2left>We decided to characterize the biobrick <a open=blank href="http://parts.igem.org/Part:BBa_K1149051">BBa_K1149051</a> (<a href=”https://2013.igem.org/Team:Imperial_College”>Imperial College London 2013</a>) using flow cytometry. By staining our cells with a Nile Red solution (0.3mg/mL of DMSO), a compound that becomes fluorescent when P3HB is present, we were able to quantify the amount of P3HB produced at the single cell level.</br></br>  
  
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<div class=text2><div class=text2left>Protocol for staining:
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The following protocol was used for staining
 
<ul><li>Thaw the samples on ice for 10 minutes</li>
 
<ul><li>Thaw the samples on ice for 10 minutes</li>
 
<li>Take 1mL of solution </li>
 
<li>Take 1mL of solution </li>
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<li>Resuspend the cells with 1 mL of ice-cold TSE Buffer</li>
 
<li>Resuspend the cells with 1 mL of ice-cold TSE Buffer</li>
 
<li>Thaw on ice for 10 minutes</li>
 
<li>Thaw on ice for 10 minutes</li>
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<li>Centrifuge for 5 minutes at 3000g at 4 degree Celsius </li>
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<li>Centrifuge for 5 minutes at 3000g at 4°C </li>
 
<li>Resuspend the cells with 1mL of ice-cold sterile water</li>
 
<li>Resuspend the cells with 1mL of ice-cold sterile water</li>
 
<li>Add 1µL of Nile Red solution Vortex the tubes</li>
 
<li>Add 1µL of Nile Red solution Vortex the tubes</li>
 
<li>Place the tubes 5 minutes in the dark </li>
 
<li>Place the tubes 5 minutes in the dark </li>
 
<li>Centrifuge for 5 minutes at 3000g at 4°C</li>
 
<li>Centrifuge for 5 minutes at 3000g at 4°C</li>
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<li>Resuspend the cells with 1mL of ice cold water and centrifuge (twice)</li></ul>
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<li>Resuspend the cells with 1mL of ice cold water and centrifuge again</li></ul>
 
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<div class=text2right><img src="https://static.igem.org/mediawiki/2017/5/51/P3HBPARISBETTENCOURT.png"><span><b>Figure 1</b>: Flow cytometer analysis of cell stained with NileRed with BBa_K1149051</span></div></div>
  
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<div class=text1>Then we used a Beckman Coulter Life Science ® flow cytometer machine to measure the intensity. We took 1 µL of every sample including a negative control (from an overnight culture of XX IDK something from Aya), and analyse them through the FL2 (575 BP filter) and FL3 (620 BP filter) channel.<div>
 
  
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<div class=text1>Flow Cytometry was used to characterize the part as we believe it is the best technique. Using fluorescence-activated cell sorting allows for the measurement of hundreds of samples each day at a minimal cost, whereas using GC/MS is not only expensive, but you can only run a few samples a day.</div>
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<div class=text1>A Beckman Coulter Life Science® flow cytometer was used to measure fluorescence at the single cell level. For each of our 2 clones, 3 replicates were performed. Our negative control consisted of stained cells that do not carry the pha operon. We took 1 µL of every sample including a negative control (from an overnight culture of wild type <i>E.Coli</i> DH5 α), and analyse them through the FL2 (575 BP filter) and FL3 (620 BP filter) channel.</br></br>
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As shown on figure 1, there is clear production of P3HB from the cells when transformed with the biobrick containing the operon. The two biological replicates have a similar behavior and have both been shown to be significantly different from the negative control (t-test, p<0.0001 in both cases).
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<div class=text1>Flow cytometry was used to further characterize the part because it allows for very accurate measurements and the study of a large amount of cells. This technique allows for the measurement of hundreds of samples each day at a minimal cost, whereas using GC/MS is not only expensive, but only a few samples can be run each day.</div>
  
 
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Latest revision as of 17:56, 12 December 2017

CONTRIBUTION

Protocol design for PHA production characterisation BBa_K1149051

We decided to characterize the biobrick BBa_K1149051 (Imperial College London 2013) using flow cytometry. By staining our cells with a Nile Red solution (0.3mg/mL of DMSO), a compound that becomes fluorescent when P3HB is present, we were able to quantify the amount of P3HB produced at the single cell level.

The following protocol was used for staining
  • Thaw the samples on ice for 10 minutes
  • Take 1mL of solution
  • Centrifuge for 5 minutes at 3000g at 4°C
  • Discard the supernatant
  • Resuspend the cells with 1 mL of ice-cold TSE Buffer
  • Thaw on ice for 10 minutes
  • Centrifuge for 5 minutes at 3000g at 4°C
  • Resuspend the cells with 1mL of ice-cold sterile water
  • Add 1µL of Nile Red solution Vortex the tubes
  • Place the tubes 5 minutes in the dark
  • Centrifuge for 5 minutes at 3000g at 4°C
  • Resuspend the cells with 1mL of ice cold water and centrifuge again
Figure 1: Flow cytometer analysis of cell stained with NileRed with BBa_K1149051
A Beckman Coulter Life Science® flow cytometer was used to measure fluorescence at the single cell level. For each of our 2 clones, 3 replicates were performed. Our negative control consisted of stained cells that do not carry the pha operon. We took 1 µL of every sample including a negative control (from an overnight culture of wild type E.Coli DH5 α), and analyse them through the FL2 (575 BP filter) and FL3 (620 BP filter) channel.

As shown on figure 1, there is clear production of P3HB from the cells when transformed with the biobrick containing the operon. The two biological replicates have a similar behavior and have both been shown to be significantly different from the negative control (t-test, p<0.0001 in both cases).
Flow cytometry was used to further characterize the part because it allows for very accurate measurements and the study of a large amount of cells. This technique allows for the measurement of hundreds of samples each day at a minimal cost, whereas using GC/MS is not only expensive, but only a few samples can be run each day.

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
bettencourt.igem2017@gmail.com