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 to create the following complete plasmid:

Our completed sensor construct utilizes the Golden Gate cloning method to avoid extra DNA bases and improve recombination accuracy [8]. The sequence begins with the restriction site BsaI, followed by a GGGG sequence, the S32-P sequence, and ended with an AAAA sequence before a second BsaI restriction site. During production, our promoter was inserted into the commercial plasmid through use of the BsaI restriction enzyme. This enzyme created a gap in the existing plasmid just before the RBS, while also creating sticky ends on our promoter sequence that would allow it to fit into this gap. The added G and A sequences in our construct were necessary to create the correct sticky ends so our promoter fits perfectly into the existing plasmid with no extraneous bases. The rest of our final plasmid came from the expression vector, the E. coli Promoter cassette, which contained a Comet GFP sequence [9] whose expression we used to quantify promoter function.

Heat sensitivity Assay

Our first assay involved producing a heat-response curve of GFP output for our S32-P and TS-Lim-P strains. We hypothesized that the rate of GFP production (quantified by rate of GFP fluorescence increase) would increase with increasing incubation temperature. Initial overnight incubations took place at 37°C to create optimal growth conditions for our E. coli and to control the cell density of each sample. Each assay included three biological replicates (cultures selected from different single transformed colonies), and three technical replicates of each of these, for nine total experimental samples from each strain. As controls, each trial also included three identical samples each of untransformed E. coli which will not fluoresce, LB+Amp media, and LB to serve as negative controls and two blanks (respectively). The LB+Amp and LB blanks allowed us to subtract any unwanted fluorescence due to the media, while simultaneous OD600 measurements allowed us to normalize GFP output to cell density. We established a four hour time course with readings every ten minutes in the plate reading, allowing enough time for protein expression to fully respond to the changing temperature. We originally ran samples for ten hours, but concluded that four hours was more than sufficient to establish the linear trend. We conducted trials at 25°C , 37°C and 42°C to characterize the heat sensitivity of the promoter at a range of temperatures.

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.

Conclusions

The key characteristic is that the GFP production rate increased as the temperature of the incubation increased, confirming the promoter’s heat-sensitivity as characterized in other papers publications. Specifically, we observed a 3.1 fold increase in GFP production rate at 42ºC over 25ºC. Interestingly, there is a linear trend, implying that at a given temperature, the σ32-inducible promoter generated relatively consistent transcription.

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