HIGH-VOLTAGE ELECTROPORATION OF BACTERIA - GENETIC-TRANSFORMATION OF CAMPYLOBACTER-JEJUNI WITH PLASMID DNA
HIGH-VOLTAGE ELECTROPORATION OF BACTERIA - GENETIC-TRANSFORMATION OF CAMPYLOBACTER-JEJUNI WITH PLASMID DNA
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DOI：
10.1073/pnas.85.3.856
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发表时间：
1988-02-01
影响因子：
11.1
通讯作者：
TOMPKINS, LS
中科院分区：
文献类型：
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作者：
MILLER, JF;DOWER, WJ;TOMPKINS, LS
电穿孔允许哺乳动物细胞和植物原生质体摄取DNA，因为它诱导细胞膜的瞬时渗透性。我们研究了利用高压电穿孔作为一种方法，通过使用肠道病原体空肠弯曲菌作为模型系统的完整的细菌细胞的遗传转化的效用。这份报告表明，应用高压放电细菌细胞允许遗传转化。我们的方法包括在质粒DNA存在下将弯曲杆菌细胞悬浮液暴露于高压指数衰减放电（5.sbd.13kV/cm）一段短暂的时间（电阻-电容时间常数2.4.sbd.26）。电转化C.空肠导致频率高达1.2 × 10 - 6。106个转化体/μ g DNA。我们研究了脉冲幅度和持续时间，细胞生长条件，二价阳离子和DNA浓度对转化效率的影响。转化子C.通过电穿孔获得的空肠在结构上含有质粒分子。此外，证据表明，C。空肠具有DNA限制和修饰系统。使用电穿孔作为一种方法，用于转化其他细菌物种和准则，其实施也进行了讨论。
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.sbd.13 kV/cm) for a brief period of time (resistance-capacitance time constant = 2.4.sbd.26 msec) in the presence of plasmid DNA. Electrical transformation of C. jejuni results in frequencies as high as 1.2 .times. 106 transformants per .mu.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 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.