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A general method for the construction of Escherichia coli mutants by homologous recombination and plasmid segregation

✍ Scribed by Kiel, J. A. K. W. ;Vossen, J. P. M. J. ;Venema, G.

Publisher: Springer
Year: 1987
Tongue: English
Weight: 904 KB
Volume: 207
Category: Article
ISSN: 0026-8925
DOI: 10.1007/bf00331592

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✦ Synopsis

A technique is presented by which mutations can be introduced into the Escherichia coli chromosome by gene replacement between the chromosome and a plasmid carrying the mutant gene. The segregational instability of plasmids in E. coli is used with high efficiency to isolate E. coli mutants. The method should be applicable to construction of mutants for any E. coli chromosomal gene provided it is dispensable, and for any E. coli strain provided it is capable of homologous recombination. The use of the method was demonstrated by constructing E. coli mutants for the glycogen branching enzyme gene (glgB) and the beta-galactosidase gene (lacZ). The results show that recombination occurs via a reciprocal mechanism indicating that the method should, in a slightly modified form, also be useful in transferring chromosomal mutations onto multicopy plasmids in vivo.

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