High-voltage electroporation of bacteria: genetic transformation of Campylobacter jejuni with plasmid DNA.
AUTOR(ES)
Miller, J F
RESUMO
Electroporation permits the uptake of DNA by mammalian cells and plant protoplasts because it induces transient permeability of the cell membrane. We investigated the utility of high-voltage electroporation as a method for genetic transformation of intact bacterial cells by using the enteric pathogen Campylobacter jejuni as a model system. This report demonstrates that the application of high-voltage discharges to bacterial cells permits genetic transformation. Our method involves exposure of a Campylobacter cell suspension to a high-voltage exponential decay discharge (5-13 kV/cm) for a brief period of time (resistance-capacitance time constant = 2.4-26 msec) in the presence of plasmid DNA. Electrical transformation of C. jejuni results in frequencies as high as 1.2 x 10(6) transformants per microgram of DNA. We have investigated the effects of pulse amplitude and duration, cell growth conditions, divalent cations, and DNA concentration on the efficiency of transformation. Transformants of C. jejuni obtained by electroporation contained structurally intact plasmid molecules. In addition, evidence is presented that indicates that C. jejuni possesses DNA restriction and modification systems. The use of electroporation as a method for transforming other bacterial species and guidelines for its implementation are also discussed.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=279654Documentos Relacionados
- Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation.
- Curing of a plasmid from E.coli using high-voltage electroporation.
- Transformation of Rhodococcus fascians by High-Voltage Electroporation and Development of R. fascians Cloning Vectors
- Resolution of antibiotic mixtures in serum samples by high-voltage electrophoresis.
- High-voltage stimulation over the human spinal cord: sources of latency variation.