In vivo transfer of chromosomal mutations onto multicopy plasmids utilizing polA strains: Cloning of an ompR 2 mutation in Escherichia coli K-12 |
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Authors: | Hannu T. Saarilahti E.Tapio Palva |
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Affiliation: | Molecular Genetics Laboratory, Department of Genetics, University of Helsinki, Arkadiankatu 7, SF-00100 Helsinki 10, Finland |
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Abstract: | Abstract We have devised a simple in vivo scheme for moving chromosomal mutations onto multicopy plasmids in Escherichia coli K-12. A plasmid clone of the relevant wild-type gene is first integrated into the chromosome of a PolA− strain carrying the desired mutation. The plasmid cointegrate formed is then resolved by P1 transduction to a PolA+ host. A certain fraction of these transductants will have the mutant allele on the plasmid. Employing this scheme we cloned an ompR 2 mutation onto a multicopy plasmid. To show that the plasmid actually contained the ompR 2 mutation, this allele was introduced back into the chromosome by the gene replacement technique of Gutterson and Koshland [1] and shown to be indistinguishable from the original ompR 2 by genetic mapping and phenotype. |
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Keywords: | In vivo cloning outer membrane porin regulation |
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