HEAT-SHOCK AND OSMOTICALLY DEPENDENT STEPS BY DNA UPTAKE AFTER ESCHERICHIA-COLI ELECTROPORATION

Currently, the major problem in the genetic transformation is to under stand how such a large molecule as the plasmid DNA passes through the cell membrane. To solve this problem we used a simplified experimental model with Escherichia coli and the plasmid pBR322: the DNA-bacteria mixture was electroporated in a sucrose solution at 0-degrees-C and at fixed electrical parameters; the samples were then directly plated in to agar. It was found that the electrically-induced bacterial transfor mation after pulsing is dependent on two factors: heat shock (DELTAT) and osmotic stress. Our results indicate that two mechanisms contribut e to these effects: (i) thermodiffusion of the DNA across the membrane and (ii) osmotic flows of the medium, containing the DNA, into the in terior of the cell.