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), that is fluorescence, we were able to quantify the amount of P3HB produced at the single cell level.</br></br></div>  
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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), 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>  
  
 
The following protocol was used for staining
 
The following protocol was used for staining
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<li>Resuspend the cells with 1mL of ice cold water and centrifuge (twice)</li></ul>
 
<li>Resuspend the cells with 1mL of ice cold water and centrifuge (twice)</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>
 
<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>

Revision as of 00:59, 2 November 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 degree Celsius
  • 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 (twice)
Figure 1: Flow cytometer analysis of cell stained with NileRed with BBa_K1149051
A Beckman Coulter Life Science ® flow cytometer machine to measure fluorescence at the single cell level. For each of our 2 clones, 3 replicates were performed and a negative control, 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 (ttest, p<0.0001 in both cases).
We chose to work with 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