Details

Autor(en) / Beteiligte

• Bernard, Philippe
• Gabarit, Philippe
• Bahassi, El Mustapha
• Couturier, Martine

Titel

Positive-selection vectors using the F plasmid ccdB killer gene

Ist Teil von

• Gene, 1994-10, Vol.148 (1), p.71-74

Ort / Verlag

Netherlands: Elsevier B.V

Erscheinungsjahr

1994

Quelle

Elsevier ScienceDirect Journals

Beschreibungen/Notizen

• 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.