Heat shock and osmotically dependent steps by DNA uptake after Escherichia coli electroporation.
Currently, the major problem in the genetic transformation is to understand 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 degree C and at fixed electrical parameters; the samples were then directly plated into agar. It was found that the electrically-induced bacterial transformation after pulsing is dependent on two factors: heat shock (delta T) and osmotic stress. Our results indicate that two mechanisms contribute to these effects: (i) thermodiffusion of the DNA across the membrane and (ii) osmotic flows of the medium, containing the DNA, into the interior of the cell.
Antonov
P A
PA
Department of Physics and Biophysics, High Medical Institute, Sofia, Bulgaria.
Maximova
V A
VA
Pancheva
R P
RP
D002462:Cell Membrane / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism*; D004058:Diffusion; D004574:Electromagnetic Fields; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics*; D006358:Hot Temperature; D009994:Osmolar Concentration; D010957:Plasmids
D004269:DNA, Bacterial