Miller J F, Dower W J, Tompkins L S
Department of Medical Microbiology, Stanford University School of Medicine, CA 94305.
Proc Natl Acad Sci U S A. 1988 Feb;85(3):856-60. doi: 10.1073/pnas.85.3.856.
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.
电穿孔法可使哺乳动物细胞和植物原生质体摄取DNA，因为它能诱导细胞膜产生瞬时通透性。我们以肠道病原体空肠弯曲菌作为模型系统，研究了高压电穿孔法作为完整细菌细胞遗传转化方法的实用性。本报告证明，对细菌细胞施加高压放电可实现遗传转化。我们的方法是在存在质粒DNA的情况下，将空肠弯曲菌细胞悬液短暂暴露于高压指数衰减放电（5 - 13 kV/cm）中（电阻 - 电容时间常数 = 2.4 - 26毫秒）。空肠弯曲菌的电转化产生的频率高达每微克DNA 1.2×10⁶个转化体。我们研究了脉冲幅度和持续时间、细胞生长条件、二价阳离子以及DNA浓度对转化效率的影响。通过电穿孔法获得的空肠弯曲菌转化体含有结构完整的质粒分子。此外，有证据表明空肠弯曲菌具有DNA限制和修饰系统。还讨论了将电穿孔法用作转化其他细菌物种的方法及其实施指南。
