Open AccessFast kinetics studies of Escherichia coli electrotransformation
Author(s) -
EYNARD Nathalie,
SIXOU Sophie,
DURAN Nathalie,
TEISSIE Justin
Publication year - 1992
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1992.tb17306.x
Subject(s) - electric field , escherichia coli , electroporation , pulse (music) , plasmid , biophysics , transformation (genetics) , chemistry , bacteria , kinetics , materials science , microbiology and biotechnology , biology , optics , physics , biochemistry , dna , genetics , gene , quantum mechanics , detector
Direct gene transfer is achieved in Escherichia coli by use of square wave electric pulsing. As observed by video monitoring, the field pulse causes bacteria to orientate parallel to the field lines. Rapid kinetic turbidity changes indicate that this process happens quickly. In these circumstances, and in pulsing conditions prone to inducing transformation, only caps are affected by the field. Considerable cytoplasmic ion leakage occurs during the pulse, affecting the interfacial ionic concentration. The pulsing‐buffer osmolarity has to be close to that used with protoplasts. Contact between the plasmid and the bacteria can be very short before the pulse but must be present during the pulse. The plasmid remains accessible to externally added DNases up to 5 days after the pulse, suggesting that the transfer step is slow. Electric‐field‐mediated transfer can be described in two steps: the anchoring process during the pulse, followed by the crossing of the membrane.