Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6Kλ origin plasmids at different copy numbers

Escherichia coli hosts were constructed for maintenance of vectors containing the y replication origin of the R6K plasmid (oriR_R6Kλ) at different copy numbers (15 or 250/cell). Such vectors require the trans-acting II protein (the pir gene product) for replication. New hosts carry pir⁺ or pir-116 on the chromosome within uidA, the E. coli gene encoding β-glucuronidase. They were made using the rep technique for allele replacement and Km^R M13ΔuidA::pir⁺ or M13ΔuidA::pir-116 phage. Because M13 cannot replicate in a rep mutant, Km^R transductants arose by integration into the chromosomal uidA locus. Segregants lacking M13 sequences (which were selected as deoxycholate-resistant (Doc^R) ones) frequently contained ΔuidA::pir⁺ or ΔuidA::pir-116 on the chromosome. In principle, this procedure could be used for the introduction of any foreign gene into any nonessential gene on the E. coli chromosome. The ΔuidA::pir⁺ and ΔuidA::pir-116 loci were subsequently transferred to a variety of E. coli strains. One such strain is a suppressor-negative one that is especially useful for transposon (Tn) mutagenesis. This strain has an integrated RP4 derivative for conjugative transfer of oriR_R6Kλ plasmids also containing oriT from RP4. In addition, new oriR_R6Kλ, oriT⁺ vectors carrying the Tc^R-encoding genes tetAR from Tn10 are described. These can be used for allele replacement by conjugative transfer of an oriR_R6Kλ, oriT⁺, tetAR plasmid containing a mutated gene into a non-p(> recipient and by subsequent selection for Tc-sensitive exconjugants.