PLASMID 35, 1–13 (1996) ARTICLE NO. 0001 Conditionally Replicative and Conjugative Plasmids Carrying lacZa for Cloning, Mutagenesis, and Allele Replacement in Bacteria WILLIAM W. METCALF, 1 WEIHONG JIANG,LARRY L. DANIELS,SOO-KI KIM, ANDREAS HALDIMANN, AND BARRY L. WANNER 2 Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907 Received August 17, 1995; revised November 14, 1995 We describe several new cloning vectors for mutagenesis and allele replacement experiments. These plasmids have the R6Kg DNA replication origin (oriR R6Kg ) so they replicate only in bacteria supplying the P 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 P. 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 lacZa for blue-white color screening of colonies for ones with plasmids carrying inserts, as well as the f1 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 Tn10) that allows for positive selection of plasmid-free segregants as tetracy- cline-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 homolo- gous 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.  1996 Academic Press, Inc. Conditionally replicative plasmids are ex- and Mekalanos, 1988) because their replica- tion depends upon the gene product of pir tremely useful for mutagenesis and allele re- placement in bacteria. By acting as suicide (encoding the P protein), which can be sup- plied in trans. Further, these plasmids can be vectors, they allow for the in vitro construc- tion of mutations in a host able to replicate maintained at low or high plasmid copy num- ber by using hosts with a chromosomal pir / them and subsequently for recombination of the mutations onto the chromosome of a host or pir-116 gene, respectively (Metcalf et al., 1994). unable to replicate them. Plasmids carrying the R6Kg DNA replication origin (oriR R6Kg ) 3 A variety of oriR R6Kg plasmids are avail- able, each with features that facilitate its use are especially useful as suicide vectors (Miller as a suicide vector (Miller and Mekalanos, 1988; Taylor et al., 1989; Donnenberg and 1 Present address: Department of Microbiology, Uni- Kaper, 1991; Hanzlik et al., 1992; Penfold versity of Illinois, Urbana, IL 61801-3704. and Pemberton, 1992; Skrzypek et al., 1993; 2 To whom correspondence should be addressed. Fax: (317) 494-0876; electronic mail: BLW@bilbo.bio.pur- Metcalf et al., 1994). In addition, many of due.edu. them carry oriT (from the plasmid RP4) so 3 Abbreviations used: oriR R6Kg , the R6Kg DNA repli- they can be conjugatively transferred to a wide cation origin; TYE, tryptone-yeast extract; LB broth, Lu- range of other bacteria from Escherichia coli ria-Bertani broth; XG, 5-bromo-4-chloro-3-indolyl-b-D- hosts that supply both pir and tra (transfer) galactopyranoside; IPTG, isopropyl b-D-thiogalactopyra- noside; TSS, tetracycline-sensitive-selective. functions in trans. However, in the absence of 1 0147-619X/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.