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.
Bernard
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P
Département de Biologie Moléculaire, Université Libre de Bruxelles, Rhode-Saint-Genèse, Belgium.
Gabant
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P
Bahassi
E M
EM
Couturier
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M
D001426:Bacterial Proteins / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D003603:Cytotoxins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005144:F Factor / Q000235:genetics*; D005822:Genetic Vectors; D008969:Molecular Sequence Data
D001426:Bacterial Proteins; D001427:Bacterial Toxins; C068356:CcdB protein, Plasmid F; D003603:Cytotoxins