Experiment 3: Discussion
Changes made for the third experiment:
- Smaller pH increments of only 0.5 pH points were tested, to more specifically determine optimal pH
- Solutions were remade such that the MgCl solution and TG salt solution used on the same cells were of a much more similar pH
- a trial at pH 9 was repeated
- More trials were done at acidic pH levels, to investigate a wider pH range
- More pBluescript was isolated, having a greater concentration than the previously isolated pBluescript
Enumeration error:
Too many CFU were too irregularly spread on amp+ plates to be counted. The plates were ranked from least (1) to most (16) CFU based on visual estimation. As the interval between ranks cannot be determined, transformation frequency cannot not be calculated and statistical tests of correlation cannot be done. The average ranking for samples of similar numbers of cells plated (200-500CFU on dilution 5 plates) were graphed. While this reduces the numbers of trials with usable data, this reduces the error caused by the inverse correlation between population density and transformation frequency. Errors in competence development for the first experiment and in serial dilution for the second experiment are present to a similar extend in the third experiment.
pH 9.3
No cells at pH 8.62-9.30 or pH 9.45-9.78 ranked higher than 9 out of 16 for the number of transformants per plate, with 16 being the most transformants. Thus, far fewer transformants at pH 8.62-9.78 were present than at pH 7.38-8.34. This decrease in transformation frequency by pH 8.62-9.78 cannot be accounted for by inverse correlation error, as fewer bacteria were plated on pH 8.62-9.78 plates than pH 7.38-8.34 plates. This suggests the transformation frequency peak at pH 9.3 in the second experiment was solely a result of error.
Optimal pH:
The third experiment suggests optimal pH is between 7.74-8.34, which agrees with Santos et al.’s findings that optimal pH is at least pH 8. The third experiment’s optimal pH range is also very similar to, but slightly more basic than Norgard’s optimal pH range. The average of optimal pH in these three experiments is pH 7.4, which supports Norgard’s optimal pH range of 7.25-7.75.
PMF involvement:
The transformation frequency did not consistently increase or decrease as components of the PMF consistently increased and decreased. There is a possibility that the pH gradient could drive transformation at extracellular pH levels more acidic than the intracellular pH, and the membrane potential could drive transformation at more basic extracellular pH levels. This would account for the decrease in frequency from pH 5.95-6.04 to pH 7.38-7.74, as pH gradient decreases in this range. This hypothesis would also account for the relatively constant frequency between pH 7.85-7.74 and pH 8.34-8.18, as membrane potential stays constant at a great magnitude within this range. However, the decrease in frequency at pH 8.62-9.78 does not support his hypothesis. It is far more likely that no component of PMF is involved. This would support the conclusions of the previous two experiments.
Why such frequent transformation in acidic conditions?
The increase in transformation frequency from pH 7.38-7.74 to pH 5.95-6.04 could be a result of errors at any stage. However, a second mechanism of transformation may operate in E. coli at acidic pH levels, which would have a different optimal pH. A different operational system has been found to become operational below pH 5.5 in the naturally transformable bacteria H. influenzae. However, this increase in transformation frequency does not agree with trends in the acidic region of the first two experiments. It is most likely that the transformation frequency increase at acidic pH levels in the third experiment is solely a result of error. The acidic range of this experiment should be repeated in further experiments for clarification.
