Factors that affect transformation of Escherichia coli with plasmid DNA

Abstract

Plasmid transformation of Escherichia coli is an essential step in many cloning experiments, yet the process is very inefficient and the action of calcium is not clear. Transformation takes place in three stages: 1) binding of DNA to the outer membrane, 2) uptake of DNA into the cell, and 3) establishment of the plasmid as a stable replicon. This study focuses on factors which influence uptake and establishment of plasmids within E. coli. Glucose, multiple hot/cold cycles, and EDTA were all tested for their effects on DNA uptake. It was observed that treatments which decrease the viability of a transformation mixture increased the proportion but the net number of transformed cells. Treatments which increase viability decrease the proportion of transformed cells. The requirement for calcium in establishment of DNA was investigated by using various chelating agents to remove calcium from the post-transformation growth medium. Removal of calcium did not affect viability of the cells, however, transformation efficiency was decreased. The results suggest that uptake of DNA cannot be improved without drastically reducing viability and the calcium may have a role in establishing of plasmids within E. coli.