Conditionally replicative and conjugative plasmids carrying lacZα for cloning, mutagenesis, and allele replacement in bacteria

We describe several new cloning vectors for mutagenesis and allele replacement experiments. These plasmids have the R6Kγ DNA replication origin (oriR(R6Kγ)) so they replicate only in bacteria supplying the Π replication protein (encoded by pir), and they can be maintained at low or high plasmid copy number by using Escherichia coli strains encoding either wild-type or mutant forms of Π. They also carry the RP4 transfer origin (oriT(RP4)) so they can be transferred by conjugation to a broad range of bacteria. Most of them encode lacZα for blue-white color screening of colonies for ones with plasmids carrying inserts, as well as the fl DNA replication origin for preparation of single stranded DNA. Particular plasmids are especially useful for allele replacement experiments because they also encode a positive counterselectable marker. One set carries tetAR (from Tn/0) that allows for positive selection of plasmid-free segregants as tetracycline-sensitive (Tet(S)) recombinants. Another set carries sacB (from Bacillus subtilis) that allows selecting plasmid-free segregants as sucrose-resistant (Suc(R)) ones. Accordingly, derivatives of these plasmids can be introduced into a non-pir host (via conjugative transfer, transformation, or electroporation), and integrants with the plasmid recombined into the chromosome via homologous sequences are selected using a plasmid antibiotic resistance marker. Plasmid- free segregants with an allele replacement can be subsequently selected as Tet(S) or Suc(R) recombinants. A number of additional features (including the presence of multiple cloning sites flanked by T3 and T7 RNA polymerase promoters) make these plasmids useful as general cloning vectors as well.