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

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.     

Selection for the transfer of phenotypically nonselectable chromosomal mutations to recombinant plasmids through introduction of an altered restriction site

We describe a simple method to select for transfer of mutant alleles from the Escherichia coli chromosome to a plasmid which formerly carried the wild-type (wt) allele. The wt allele on the plasmid is modified by introduction of a unique restriction site (e.g., XhoI) and transformed into a rec ⁺ strain carrying the mutant allele on the chromosome. Upon homogenotization, the efficiency of which was increased by UV irradiation of the transforming plasmid [Chattoraj et al., Gene 27 (1982) 213–222], plasmids carrying the mutant allele are formed which are resistant to XhoI. These plasmids are selected from the population by resistance to XhoI digestion coupled with the low transformation efficiency of linear DNA molecules in recA⁻ strain. The method is efficient and rapid and has particular advantages in situations where the mutant allele is difficult to detect by its phenotype.