Part:BBa_M50018:Experience

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Applications of BBa_M50018

This part was inserted into an expression vector plasmid by DNA 2.0 containing GFP, so we wanted to develop a heat-response curve quantifying GFP output. After transforming this plasmid into chemically competent E. coli, we selected colonies based on ampicillin resistance and produced 3 liquid cultures of biological replicates. 3 wells each of these cultures (for 9 total samples) were held in a spectrophotometer/plate reader while shaking for 4 hours, with the machine taking readings of CometGFP fluorescence and cell density (OD600) every 10 minutes). After normalizing fluorescence to cell density, we established the following curves at 25ºC, 37ºC, and 42ºC:

These graphs indicate that the slope of our observed GFP readings increased with temperature. For the S32-P bacteria, the rate of GFP production started at an increase of 1.84 arbitrary units/minute at 25°C, 2.21 at 37°C and finally 6.05 at 42°C. For the TS-Lim-P cultures, rates increased from 2.65 at 25°C to 3.66 at 37°C to 8.30 at 42ºC. This data demonstrates that there was a significant increase in rate of GFP production as temperature increased, which asserts our hypothesis that the σ32 promoter creates heat-sensitive protein expression. The fluorescence always increased linearly over time, which is supported by the high R2 values of our lines of best fit, all of which are near or above 0.9. As expected, the E.Coli control had little fluorescence resulting in a relatively flat line of best fit, and indicating a GFP production rate close to zero.

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