CONDITIONALLY REPLICATIVE AND CONJUGATIVE PLASMIDS CARRYING LACZ-ALPHA FOR CLONING, MUTAGENESIS, AND ALLELE REPLACEMENT IN BACTERIA

We describe several new cloning vectors for mutagenesis and allele rep lacement experiments. These plasmids have the R6K gamma DNA replicatio n origin (oriR(R6K gamma)) so they replicate only in bacteria supplyin g the Pi replication protein (encoded by pir), and they can be maintai ned at low or high plasmid copy number by using Escherichia coli strai ns encoding either wild-type or mutant forms of Pi. They also carry th e RP4 transfer origin (oriT(RP4)) SO they can be transferred by conjug ation to a broad range of bacteria. Most of them encode lacZ alpha for blue-white color screening of colonies for ones with plasmids carryin g inserts, as well as the fl DNA replication origin for preparation of single-stranded DNA. Particular plasmids are especially useful for al lele replacement experiments because they also encode a positive count erselectable marker. One set carries tetAR (from Tn10) that allows for positive selection of plasmid-free segregants as tetracycline-sensiti ve (Tet(S)) recombinants. Another set carries sacB (from Bacillus subt ilis) that allows selecting plasmid-free segregants as sucrose-resista nt (Suc(R)) ones. Accordingly, derivatives of these plasmids can be in troduced 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 r eplacement can be subsequently selected as Tet(S) or Suc(R) recombinan ts. A number of additional features (including the presence of multipl e cloning sites flanked by T3 and T7 RNA polymerase promoters) make th ese plasmids useful as general cloning vectors as well. (C) 1996 Acade mic Press, Inc.