Difference between revisions of "Part:BBa M50073:Experience"

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Applications of BBa_M50073

Upon receiving our plasmids from DNA 2.0, we transformed pPink and pTurquoise into E. coli competent cells, which were obtained commercially. Using the heat-shock transformation protocol, we transformed three separate E. coli cells; one with pPink, one with pTurquoise, and one with both pPink and pTurquoise. Since we did not know the concentration of our plasmids from DNA 2.0, we initially used 1 µl of DNA for our single transformations, and 1 µl of each for our double-transformation.

Following incubation, we observed several colonies on both our pPink and pTurquoise plates indicating a successful transformation, but few colonies for our double-transformation. We repeated the transformation protocol detailed above for the double plasmids, and added 2 µl of both the plasmids in an effort to get more DNA into the cells. This transformation yielded a much greater number of colonies, indicating that our transformation was surely a success! See Figure 3 below for confirmation of successful transformations. We inoculated single colonies from these plates in liquid LB media with their respective antibiotic, and then allowed them to grow overnight in a 37°C shaking incubator. These cultures were then made into glycerol freezer stocks. In 2D barcode tubes we added 500 µl of 50% glycerol, and 500 µl of liquid culture. We created a glycerol stock for each our pPink, pTurquoise, and pPink + pTurquoise strains, logged them into the BIOE44 system, and stored them in the -80°C freezer for future use.

Experiment 1: Testing pPink and pTurquoise Functionality at Different pHs

With our successfully transformed plasmids, we wanted to test our device’s ability to produce pink and turquoise fluorescent proteins in response to different pHs. Since the documentation of our promoters indicates that they are sensitive for pH ranges 4.8 to 7 for pPink, and 5.5 to 8 pH for pTurquoise, we wanted to test each transformed strain within the overall pH range of 4.8 to 8. We chose this pH range based on literature, which has demonstrated E. coli growth between pH’s 4 and 8.¹⁰ We did not stray from this range in order to ensure optimal conditions for cell growth and eliminate extraneous factors that could affect our fluorescence results.

We prepared LB media stocks for pHs: 4.8, 5, 5.5, 6, 6.6, 7, 7.5, and 8, to test how our plasmids respond to incremental pH changes. We poured 100 mL of liquid LB media into bottles over flame, and then adjusted their pH by adding .1 M HCl or 10% NaOH dropwise. We then autoclaved all of the bottles on liquid for 15 minutes in order to ensure sterility of our media. Using these pH-media stocks, we then made 10 mL antibiotic-media stocks at each pH.

To set up our pH-fluorescence assay, we first measured the OD600 of our cultures: pPink, pTurquoise, pPink + pTurquoise, and pHek, which served as our negative control to ensure a low starting OD of 0.01 A. We set up our pH-experiment in two 96, 1 mL well plates by adding our prepared antibiotic-media for each pH, and then using normal LB + antibiotic media for a positive control. Triplicates of each experimental strain, pPink, pTurquoise, and pPink + pTurquoise, were plated along with the negative control pHek and a blank of LB media. Reference the Supplemental Information section for the experimental plate map executed.

These experimental cultures were allowed to grow in the 37°C shaking incubator for two days, and then 200 µl of each sample was pipetted into a 96, 200 µl well plate for measurement by the plate reader. For each sample, we measured the OD600, pink fluorescence with excitation/emission of 553/592 nm, and turquoise fluorescence with excitation/emission of 434/474 nm. The fluorescence values were normalized by the OD, and are displayed below in Figures 2 and 3.

Our pink fluorescence assays demonstrate that the pPink promoter is highly sensitive at pH 4.8, as we observe a high spike in fluorescence at 4.8 and decrease at higher pHs. On the other hand, our measurements for pTurquoise reflect much lower pH-specificity, as we observe fairly high fluorescence values for the majority of pH ranges in both pTurquoise and pPink + pTurquoise. Although we do observe a slight spike in both transformed species around pH 6, our data leads us to believe that our pTurquoise promoter is generally leaky. The low fluorescence measured in our negative control, pHek, supports the experimental efficacy in our results.

Experiment 2: Observing how pPink and pTurquoise Function Over Time

Next, we wanted to repeat our initial experiment, and obtain time-point data in order better understand how our device functions overtime. Our time-point experiment gave us kinetic information that would elucidate how quickly or slowly our promoters were reacting to changes in pH, which is important information for practical usage of the device. We followed a similar experimental setup as described above, using the LB-pH media stocks with antibiotic we had previously prepared. However for this experiment, we plated our cultures in a a 96, 200 µl well plate as the cultures would be growing inside the plate reader. Reference the Supplemental Information section for the experimental plate map executed. We preprogrammed the plate reader to shake and maintain a constant temperature of 37°C. The plate reader was also programmed to measure the OD600, pink fluorescence, and turquoise fluorescence every 15 minutes over the course of 24 hours. The results of our experiment are displayed below in Figures 6, 7, and 8; reference the Supplemental Information section for more data on pTurquoise protein production.

Our results may suggest that the acid shock response promoter used in pPink is activated by pH sometime after 24 hours. Alternatively, perhaps the low copy number ORI in this plasmid prevents promoter activity from occurring within 24 hours. An interesting future experiment would be to conduct a time-point experiment for 48 hours to determine at what time the promoter is activated. pTurquoise exhibited high levels of protein production from pH of 4.8 to 7.5, with a slight peak around pH 6; we observed similar trends for pTurquoise over 24 hours.

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