Differential Transformation Efficiencies Observed for pUC19 and pBR322 in E. coli May Be Related to Calcium Chloride Concentration

Calcium chloride is commonly included in buffers used to generate chemocompetent bacterial cells. The mechanism that underlies the uptake of exogenous DNA into bacteria is not completely understood. Previous studies have suggested that calcium ions may function to neutralize electrostatic repulsion between DNA and membrane proteins, thereby allowing exogenous DNA to enter membrane pores. Other studies have shown that transformation efficiency decreases as plasmid size increases. We hypothesized that increasing calcium chloride concentrations during the transformation may increase transformation efficiency of larger sizes. To test whether or not calcium chloride concentration is related to differential transformation efficiency of plasmids of different sizes, we compared the transformation efficiency of pUC19 (2686 base pairs) and pBR322 (4361 base pairs) at varying calcium chloride concentrations of 0.05 M, 0.1 M, 0.15 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M and 1.0 M. We observed maximum transformation efficiencies of pUC19 and pBR322 at 0.15 M CaCl2 and 0.1 M CaCl2, respectively. Further increases in calcium chloride concentrations resulted in a decrease in transformation efficiency for both plasmids. Above 0.2 M CaCl2, no transformants were observed with either plasmid. A decreasing trend in cell viability was observed in the absence of antibiotic suggesting that the increasing hypertonic environment may affect cell viability. Based on our experiments, maximal transformation efficiency for pUC19 was found to be 4.8x10 colony forming units per ug at 0.15 M of calcium chloride while pBR322 had a maximum transformation efficiency of 1.8x10 colony forming units per ug at 0.1 M of calcium chloride. Taken together, our results do not show a clear relationship between calcium chloride concentration and plasmid size with respect to transformation efficiency.