High-Voltage Electroporation of Bacteria: Genetic Transformation of Campylobacter jejuni with Plasmid DNA

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 85; no. 3; pp. 856 - 860
• Main Authors: Miller, Jeff F., Dower, William J., Tompkins, Lucy S.
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01.02.1988; National Acad Sciences
• Subjects: Bacteriological Techniques; Bacteriology; Biological and medical sciences; Campylobacter; Campylobacter fetus - genetics; Campylobacter jejuni; Capacitors; Cell Membrane Permeability; DNA; DNA, Bacterial - genetics; DNA, Recombinant; Electric field strength; electric fields; Electric potential; Electric pulses; Electroporation; Field strength; Fundamental and applied biological sciences. Psychology; Genetic Techniques; Genetics; membrane permeability; Microbiology; Plasmids; Time constants; transformation; Transformation, Bacterial; Plasmid; Electric field; Genetic transformation; Spirillales; DNA; Pulsed field; Spirillaceae; Bacteria; Campylobacter jejuni; Genetic transfer
• DOI: 10.1073/pnas.85.3.856
• ISSN: 0027-8424

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 × 106 transformants per μ g 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.