Part:BBa_M50018:Experience
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
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 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.
User Reviews
UNIQc228a96b49567d9e-partinfo-00000000-QINU UNIQc228a96b49567d9e-partinfo-00000001-QINU