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

FEBS Letters

Lange, Hans¹  Lemke, Hilmar¹  Deyev, Sergey²  Mokros, Thilo¹  Yazynin, Sergey¹ 

[1] Biochemisches Institut der Medizinischen Fakultät der Christian-Albrechts-Universität zu Kiel, Olshausenstrasse 40, 24118 Kiel, Germany;Engelhardt Institute for Molecular Biology, Russian Academy of Sciences, Vavilov str.32, 117984 Moscow, Russia

关键词: Barnase;    Barstar;    Cloning;    Ribonuclease;   

DOI  :  10.1016/S0014-5793(99)00661-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A new phagemid cloning vector for positive selection of recombinants, pBa-7, was constructed which contains an active barnase gene encoding the cytotoxic ribonuclease from Bacillus amyloliquefaciens, under control of the lac promoter. PBa-7 is a derivative of the high-copy number pBluescript II KS+ phagemid in which the modified barnase killer gene has been fused downstream from the lac promoter of the pBluescript II KS+ multiple restriction site. When a lacI^q-negative Escherichia coli strain is transformed by this vector, the active barnase blocks bacterial growth by massive RNA destruction [1]. However, if barnase is inactivated by insertion of a foreign DNA fragment into the multirestriction site of the vector, this recombinant plasmid no longer interferes with the host viability. The positive selection of recombinant clones is highly efficient and bench manipulations are considerably simplified. When E. coli transformants are plated out on rich medium with ampicillin, only cells containing recombinant plasmids give rise to colonies. 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 lacI^q-positive E. coli hosts. Hence, pBa-7 seems to be suitable for most genetic engineering manipulations.

【 授权许可】

Unknown