Abstract
| Original language | English |
|---|---|
| Pages (from-to) | 351-354 |
| Number of pages | 4 |
| Journal | FEBS Letters |
| Volume | 452 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 11 Jun 1999 |
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Keywords
- Barnase
- Barstar
- Cloning
- Ribonuclease
Cite this
A new phagemid vector for positive selection of recombinants based on a conditionally lethal barnase gene. / Yazynin, Sergey; Yazynin, Sergey; Lange, Hans; Mokros, Thilo; Deyev, Sergey; Lemke, Hilmar.
In: FEBS Letters, Vol. 452, No. 3, 11.06.1999, p. 351-354.Research output: Contribution to journal › Article
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TY - JOUR
T1 - A new phagemid vector for positive selection of recombinants based on a conditionally lethal barnase gene
AU - Yazynin, Sergey
AU - Yazynin, Sergey
AU - Lange, Hans
AU - Mokros, Thilo
AU - Deyev, Sergey
AU - Lemke, Hilmar
PY - 1999/6/11
Y1 - 1999/6/11
N2 - 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. Copyright (C) 1999 Federation of European Biochemical Societies.
AB - 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. Copyright (C) 1999 Federation of European Biochemical Societies.
KW - Barnase
KW - Barstar
KW - Cloning
KW - Ribonuclease
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U2 - 10.1016/S0014-5793(99)00661-4
DO - 10.1016/S0014-5793(99)00661-4
M3 - Article
VL - 452
SP - 351
EP - 354
JO - FEBS Letters
JF - FEBS Letters
SN - 0014-5793
IS - 3
ER -