Team:Paris Bettencourt/Contribution

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@iGEM_Paris

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), this technique of Fluorescence-activated cell sorting allowed us to measure the intensity of the Nile Red stained cell containing P3HB.

Protocol 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)

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 wild type E.Coli DH5 α), and analyse them through the FL2 (575 BP filter) and FL3 (620 BP filter) channel.

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.

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