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), 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.