DNA transfection of Escherichia coli by electroporation

Taketo, Akira

doi:10.1016/0167-4781(88)90158-3

Cited by 107 publications

(60 citation statements)

References 18 publications

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“…The surface-diffusion step described by Tsong [ 141 is then supposed to rollow. Another limitation to Tsong's proposal is that, in a pulsing buffer with 1 mM MgC12, DNA must be present during the pulse [24].…”

Section: Discussionmentioning

confidence: 99%

Fast kinetics studies of Escherichia coli electrotransformation

Eynard

Sixou

Durán

et al. 1992

European Journal of Biochemistry

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Direct gene transfer is achieved in Escherichiu coli by use of square wave electric pulsing. As observed by vidco 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 intcrfacial 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.The organization and properties of cell membranes can be altcred when the membrane-potential difference is brought to a critical value. This was shown by electropulsation for the first time in 1972, when the permeability of chromaffin granules was reversibly increased when vesicles were submitted to short high-intensity electric pulses (so called electropermeabilization) [I], a property which has since been observed in many cell systems. In 1982, gene transfer was mediated in eukaryotic cells by electropulsation (electrotransformation) [2] and gene expression was obtained showing genomic integration of the plasmid. Electrotransformation was successfully applied to many systems of mammalian cells and plant protoplasts [3]. More recently, gene transfer was obtained with walled systems, such as bacteria [4] or yeasts [5], and electrotransformation is, in [act, now routinely used in many bacterial-genetics laboratories. Yiclds as high as 20 -30% have been reported using strains which were known to be casily transformed by the CaC1, method [6 -1 I]. Nevertheless, mechanisms of electrotransformation remain unexplained in the case of bacteria, as with other systems. Optimization of thc procedure remains, in most cases, empirical. A correlation has nevertheless been shown by our group between electropermeabilization and electrotransformation in the case of Escherichiu coli and Salmonella thyphimurium [12].However, a lack of knowledge of the basic processes remains the major handicap to the definition of efficient transformation conditions. Several processes are induced at the cell level upon electropulsation. Thc well-known Joule-heating phenomenon cannot be neglected under electrical conditions inducing electrotransformation. Dipolcs are induced by the electric field and are oriented by a field effect and reorientation of the Corre.pmdence to J. Teissit, Centre de Recherche dc Biochimie et de Gtnetique Ccllulaires du Centre National de la Recherche Scieiitifiquc, 118 route de Narbonne, F-31062 Toulouse. Cedex, France pulsed vesicles may occur. The most dramatic...

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Section: Discussionmentioning

confidence: 99%

Fast kinetics studies of Escherichia coli electrotransformation

Eynard

Sixou

Durán

et al. 1992

European Journal of Biochemistry

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“…Originally developed for transfection of eukaryotic cells 1 , electroporation was subsequently adapted for transformation of E. coli 2,3 . In bacteriology, electroporation has become the laboratory standard and has been modified to transform a wide range of bacteria including Gram-negative Pseudomonas 4 , Salmonellae 5 , Vibrios 6 , Serratiae, and Shigellae…”

Section: Introductionmentioning

confidence: 99%

Rapid Protocol for Preparation of Electrocompetent <em>Escherichia coli</em> and <em>Vibrio cholerae</em>

Gonzales¹,

Brooks

Pukatzki

et al. 2013

JoVE

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Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

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“…Electroporation, as a gene delivery strategy, initially comprised the combination of naked DNA injection and pulsed electric field treatment (29,30). The success of in vitro delivery by electroporation has led to the development of in vivo applications (31 -33).…”

Section: Discussionmentioning

confidence: 99%