Calcium Chloride Made E. coli Competent for Uptake of Extraneous DNA Through Overproduction of OmpC Protein |
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Authors: | Pulakesh Aich Monobesh Patra Arijit Kumar Chatterjee Sourav Singha Roy Tarakdas Basu |
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Institution: | 1. Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, 741 235, West Bengal, India 2. Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India
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Abstract: | In the standard method of transformation of Escherichia coli with extraneous DNA, cells are made competent for DNA uptake by incubating in ice-cold 100?mM CaCl2. Analysis of the whole protein profile of CaCl2-treated E. coli cells by the techniques of one- and two-dimensional gel electrophoresis, MALDI-MS and immunoprecipitation revealed overproduction of outer membrane proteins OmpC, OmpA and heat-shock protein GroEL. In parity, transformation efficiency of E. coli ompC mutant by plasmid pUC19 DNA was found to be about 40?% lower than that of the wild type strain. Moreover, in E. coli cells containing groEL-bearing plasmid, induction of GroEL caused simultaneous overproduction of OmpC. On the other hand, less OmpC was synthesized in E. coli groEL mutant compared to its wild type counterpart, by CaCl2-shock. From these results it can be suggested that in the process of CaCl2-mediated generation of competence, the heat-shock chaperone GroEL has specific role in DNA entry into the cell, possibly through the overproduced OmpC and OmpA porins. |
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