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Role of membrane potential on artificial transformation of E. coli with plasmid DNA

Paper ID Volume ID Publish Year Pages File Format Full-Text
25555 43581 2006 7 PDF Available
Title
Role of membrane potential on artificial transformation of E. coli with plasmid DNA
Abstract

The standard method of transformation of Escherichea coli with plasmid DNA involves two important steps: cells are first suspended in 100 mM CaCl2 at 0 °C (in which DNA is added), followed by the administration of a heat-pulse from 0 to 42 °C for 90 s [Cohen, S., Chang, A., Hsu, L., 1972. Nonchromosomal antibiotic resistance in bacteria. Proc. Natl. Acad. Sci. U.S.A., 69, 2110–2114]. The first step makes the cells competent for uptake of DNA and the second step is believed to facilitate the DNA entry into the cells by an unknown mechanism. In this study, the measure of membrane potential of the intact competent cells, at different steps of transformation process, either by the method of spectrofluorimetry or that of flow cytometry, indicates that the heat-pulse step (0 → 42 °C) heavily decreases the membrane potential. A subsequent cold shock (42 → 0 °C) raises the potential further to its original value. Moreover, the efficiency of transformation of E. coli XL1 Blue cells with plasmid pUC19 DNA remains unaltered when the heat-pulse step is replaced by the incubation of the DNA-adsorbed competent cells with 10 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 90 s at 0 °C. Since the CCCP, a well-known protonophore, reduces membrane potential by dissipating the proton-motive-force (PMF) across E. coli plasma membrane, our experimental results suggest that the heat-pulse step of the standard transformation procedure facilitates DNA entry into the cells by lowering the membrane potential.

Keywords
E. coli; Plasmid DNA; Artificial transformation; Membrane potential
First Page Preview
Role of membrane potential on artificial transformation of E. coli with plasmid DNA
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 127, Issue 1, 15 December 2006, Pages 14–20
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering