The Effect of Spheroplast Formation on the Transformation Efficiency in Escherichia coli DH5α

Abstract

Based on the observation that transformation in Escherichia coli is an inefficient process, it was hypothesized that transformation may be hindered by the presence of the outer membrane and the cell wall. To test this hypothesis, cells which lack both a cell wall and most of its outer membrane, termed spheroplasts, were created and transformed with pUC8 plasmid. Spheroplast formation was induced by incubating exponentially growing E. coli DH5 α in ampicillin and sucrose. To transform these spheroplasts, the cells were subjected to a modified version of the Hanahan protocol, which involves treating the cells with calcium chloride. The results of this experiment suggest that spheroplasts can -4 transform at 4.9 × 10. This is 100 fold more efficient than using the modified Hanahan protocol on wildtype E. coli DH5 α cells.