In vivo transfer of chromosomal mutations onto multicopy plasmids utilizing polA strains: Cloning of an ompR 2 mutation in Escherichia coli K-12

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.