Difference between revisions of "Part:BBa K1149051:Experience"

Edinburgh OG 2018

The iGEM Edinburgh OG 2018 team decided to characterize the ideal glucose concentration for Hybrid promoter with PHA operon

E. coli cells were cultured with different concentrations of glucose at 37 ℃ and optical density was measured at cultivation time of 6 hours, 24 hours, 30 hours 48 hours, 56 hours and 72 hours.

Figure 1 Comparison of growth curve of recombinant E. coli harbouring pSB1C3-phaCAB for different concentration of glucose.

The iGEM Edinburgh OG 2018 team decided to assess the Real-time PHA detection by Semi-quantitative analysis (Nile Red Fluorescent intensity-through using Image J program)

Cell cultures were harvested at different cultivation hours including 6 hours, 24 hours, 30 hours and 48 hours. The bars in blue and orange represented signal intensity of cells harboured pSB1C3-phaCAB and pSB1C3 respectively. The bars in yellow represented signal intensity resulting from PHA, which were calculated from the difference between intensity of pSB1C3-phaCAB and intensity of pSB1C3.

Figure 2. Fluorescent intensity of cells harboured pSB1C3 or pSB1C3-phaCAB at different cultivation time.

The team then decided To determine the influence of glucose availability on PHA production level, in addition to the growth curves with different glucose concentrations.

Figure 3. The fluorescent intensity of PHA produced with different glucose concentrations. (48 hours cultivation). Error bars represented standard deviations.

Effect of different concentration of propionic acid. In order to determine the tolerance of higher concentration of propionic acid, E. coli harbouring pSB1C3-phaCAB plasmids were cultured with 3 % glucose and different concentration of propionic acid for 60 hours.

Figure 4. Comparison of growth curves with different concentration of propionic acid. E. coli harbouring pSB1C3-phaCAB was cultivated with different glucose and propionic acid. The time of adding propionic acid was pointed out by red arrow.

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The iGEM Paris Bettencourt 2017 team decided to characterize the biobrick BBa_K1149051 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)

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.