Initial stage of DNA-electrotransfer into E. coli cells.
The mechanism of electrotransfer of DNA into Escherichia coli cells was investigated under conditions optimal for genetic transformation or transfection. Simple mixing in 10% polyethylene glycol 6000 did not cause binding of DNA to the recipient bacteria. When subjected to a high electric field, however, 90-98% of the input plasmid or phage DNAs were complexed with the cells. By application of the electric field, a significant amount of biotin-labeled DNA was bound onto the recipient surface, as detected by fluorescein isothiocyanate coupled avidin. When subjected to a high voltage pulse, DNA molecules were rapidly attracted toward the anode. Concurrently, the electric field induced the orientation of bacterial cells, along the field lines and their movement toward the anode. Since the bacterial movement was relatively slow, a substantial fraction of DNA molecules must strike the cathode-facing end or side of the recipient cells. Irrespective of the high efficiency of DNA transformation, the voltage pulse did not induce release of alkaline phosphate and beta-galactosidase. The electrotransferred DNA first remained sensitive to Tris-EDTA treatment, and became refractory to spheroplasting only after incubation at 37 degrees C. These results indicate that the infecting DNA is electrophoretically plugged to the outer membrane loosened by the voltage pulse.
Kimoto
H
H
Department of Biochemistry I, Fukui Medical School.
Taketo
A
A
D002463:Cell Membrane Permeability; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D018274:Electroporation / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D018014:Gene Transfer Techniques; D014162:Transfection / Q000379:methods
D004247:DNA