Open AccessIn vivo transfer of chromosomal mutations onto multicopy plasmids utilizing polA strains: Cloning of an ompR 2 mutation in Escherichia coli K‐12
Author(s) -
Saarilahti Hannu T.,
Palva E.Tapio
Publication year - 1985
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1985.tb01560.x
Subject(s) - plasmid , genetics , biology , mutant , escherichia coli , mutation , chromosome , gene , microbiology and biotechnology , clone (java method) , cloning (programming) , computer science , programming language
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.