A new phagemid vector for positive selection of recombinants based on a conditionally lethal barnase gene

A new phagemid cloning vector for positive selection of recombinants, pBa-7 , was constructed which contains an active barnase gene encoding the cytoto xic ribonuclease from Bacillus amyloliquefaciens, under control of the Inc promoter. PBa-7 is a derivative of the high-copy number pBluescript II KSphagemid in which the modified barnase killer gene has been fused downstrea m from the lac promoter of the pBluescript II KS+ multiple restriction site . When a lacI(q)-negative Escherichia coli strain is transformed by this ve ctor, the active barnase blocks bacterial growth by massive RNA destruction [1], However, if barnase is inactivated by insertion of a foreign DNA frag ment into the multirestriction site of the vector, this recombinant plasmid no longer interferes with the host viability. The positive selection of re combinant clones is highly efficient and bench manipulations are considerab ly simplified. When E. coli transformants are plated out on rich medium wit h ampicillin, only cells containing recombinant plasmids give rise to colon ies. In a lacI(q)-positive host, the positive selection is IPTG-dependent, Therefore, pBa-7 phagemid can be amplified and prepared in large quantities from lacl(q)-positive E. coli hosts, Hence, pBa-7 seems to be suitable for most genetic engineering manipulations, (C) 1999 Federation of European Bi ochemical Societies.