Experiment 1: Discussion
Variation in pH Levels of Solutions
The pH levels of solutions varied slightly from that of the buffers when used for incubation at 0oC, due to the different ionization constant of buffer acid at different temperatures. The pH of magnesium chloride solutions and TG salts solutions used on the same cells also differed slightly due to differing solutions compositions. For experiment 3, magnesium chloride and TG salt solutions were remade to be of much more similar pH levels. The actual pH levels of the solutions at 0oC, not the pH of the buffers, were used for all data analysis.
Serial Dilution Error
The great variation in the calculated number of cells plated on each amp+ plate, despite the similar initial number of cells in each sample, suggests a major error in the serial dilution process. The transformation frequencies vary inversely with the number of cells plated on each amp+ plate, suggesting that transformation frequencies reflect a significant degree of error in determining the total number of bacteria present. The limited similarity between the graph of the number of transformants per amp+ plate and transformation frequency supports this interpretation. Vortexing of each serial dilution stage, as was done in this experiment, was later observed to unevenly resuspend cells. Uneven mixing would severely alter the actual dilution factor of the cells, and calculations of both the number of cells plated and transformation frequency.
Optimal pH
The only trend in transformation frequency not solely accounted for by these errors is the peak at pH 6.88-7.14. This is similar to Norgard’s findings that transformation frequency is greatest at pH 7.25-7.75. While transformation frequency could increase further between pH 7.14 and 7.88, at which no E. coli were transformed in this experiment, the extremely low transformation frequency at pH 7.88 suggests the optimum pH range does not extend to pH 7.75. Contrary to this experiment, Norgard found transformation frequency is greater at pH 8 than pH 7.35
PMF involvement
This experiment does support Sabelnikov and Domaradsky’s conclusion that neither component of the PMF is involved in E. coli transformation. Transformation frequency increases up to pH 7, as ΔΨ increases and ΔpH decreases, yet transformation frequency decreases by pH 7.88, when ΔΨ continues to increase and ΔpH becomes constant.36 Although both the transformation frequency and the number of transformants per plate dropped by pH 7.88, suggesting that this drop is not solely a reflection of serial dilution error, the standard deviation of the number of transformants per plate is very large at pH 7.88. Therefore, this drop could solely be a result of error.
Competence Development Error
Major error was involved in making the cells competent. Slightly different volumes of bacteria were initially grown, the concentrations of solutions varied slightly, different quantities of cells would have been lost with decanted supernatants, samples were resuspended to slightly different degrees, and different samples spent slightly different lengths of time in different solutions. These errors affect the degree of competency of different cell samples, both directly and through altering the concentration of suspended cells.37 These errors impact all data except that used in calculating the number of cells plated. A greater number of transformants per pH tested would more clearly reflect any relationship between pH and transformation frequency. The troubleshooting test was performed to determine how the transformation frequency could be increased the most.
