Positive-selection vectors using the F plasmid ccdB killer gene.

Plasmids pKIL18/19 are positive-selection cloning vectors containing an active cytotoxic ccdB gene under the control of the lacP promoter. They are derivatives of high-copy-number pUC18/19 plasmids in which the ccdB killer gene has been fused in phase downstream from the lacP MCS18 and MCS19 multiple cloning sites. When an Escherichia coli wild-type gyrA+ strain is transformed by such vectors, the ccdB gene product blocks bacterial growth. However, if ccdB is inactivated by insertion of a foreign DNA fragment, this recombinant plasmid no longer interferes with host viability. The positive selection of recombinant clones is highly efficient and bench manipulations are simplified to the utmost: E. coli transformants are plated on rich medium and only cells containing recombinant plasmids give rise to colonies. The CcdB protein is a potent poison of gyrase and the gyrA462 mutation confers total resistance to CcdB [Bernard and Couturier, J. Mol. Biol. 226 (1992) 735-745]. Therefore, pKIL18/19 vectors can be amplified and prepared in large quantities in a gyrA462 host. Like pUC vectors, pKIL vectors are designed for general cloning/sequencing procedures.

[1]  J. Rosner.  Formation, induction, and curing of bacteriophage P1 lysogens. , 1972, Virology.

[2]  L. Wyns,et al.  The F plasmid CcdB protein induces efficient ATP-dependent DNA cleavage by gyrase. , 1993, Journal of molecular biology.

[3]  T. Miki,et al.  Control of cell division by sex factor F in Escherichia coli. I. The 42.84-43.6 F segment couples cell division of the host bacteria with replication of plasmid DNA. , 1984, Journal of molecular biology.

[4]  H. Boyer,et al.  Positive-selection vectors utilizing lethality of the EcoRI endonuclease. , 1986, Gene.

[5]  M. Couturier,et al.  Ham22, a mini‐F mutation which is lethal to host cell and promotes recA‐dependent induction of lambdoid prophage. , 1983, The EMBO journal.

[6]  M. Couturier,et al.  Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes. , 1992, Journal of molecular biology.

[7]  B. Henrich,et al.  Use of the lysis gene of bacteriophage phi X174 for the construction of a positive selection vector. , 1986, Gene.

[8]  G. Pósfai,et al.  The construction of a versatile plasmid vector that allows direct selection of fragments cloned into six unique sites of the cI gene of coliphage 434. , 1984, Gene.

[9]  G. Warren,et al.  Lethality of palindromic DNA and its use in selection of recombinant plasmids. , 1982, Gene.

[10]  J. Vijg,et al.  Application of galactose-sensitive E. coli strains as selective hosts for LacZ- plasmids. , 1992, Nucleic acids research.

[11]  I. Beacham,et al.  Positive selection vectors: a small plasmid vector useful for the direct selection of Sau3A-generated overlapping DNA fragments. , 1984, Gene.

[12]  S. Falkow,et al.  Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. , 1977, Gene.

[13]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.

[14]  D. Dean.  A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids. , 1981, Gene.

[15]  B. Müller-Hill,et al.  Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[16]  M. Couturier,et al.  Lon‐dependent proteolysis of CcdA is the key control for activation of CcdB in plasmid‐free segregant bacteria , 1994, Molecular microbiology.

[17]  Arakawa Yoshichika,et al.  Construction of a novel suicide vector: selection for Escherichia coli HB101 recombinants carrying the DNA insert. , 1991 .

[18]  G. Humphreys,et al.  Expression of the Eco RI restriction-modification system and the construction of positive-selection cloning vectors. , 1982, Gene.

[19]  T. Ogura,et al.  Effects of the ccd function of the F plasmid on bacterial growth , 1985, Journal of bacteriology.

[20]  D. Hanahan.  Studies on transformation of Escherichia coli with plasmids. , 1983, Journal of molecular biology.

[21]  J. Sadler,et al.  Plasmids containing many tandem copies of a synthetic lactose operator. , 1980, Gene.

[22]  B. Sauer,et al.  A positive selection vector for cloning high molecular weight DNA by the bacteriophage P1 system: improved cloning efficacy. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[23]  T. Roberts,et al.  A plasmid cloning vehicle allowing a positive selection for inserted fragments. , 1980, Gene.