Abstract:
: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 lacIq-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 lacIq-positive host, the positive selection is IPTG-dependent. Therefore, pBa-7 phagemid can be amplified and prepared in large quantities from lacIq-positive E. coli hosts. Hence, pBa-7 seems to be suitable for most genetic engineering manipulations.
journal_name
FEBS Lettjournal_title
FEBS lettersauthors
Yazynin S,Lange H,Mokros T,Deyev S,Lemke Hdoi
10.1016/s0014-5793(99)00661-4keywords:
subject
Has Abstractpub_date
1999-06-11 00:00:00pages
351-4issue
3eissn
0014-5793issn
1873-3468pii
S0014-5793(99)00661-4journal_volume
452pub_type
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