A novel vector for direct cloning PCR fragments by positive selection based on the lethal barnase.
A novel vector for direct PCR fragments cloning by positive selection, pBN, was constructed based on the lethal barnase from Bacillus amyloliquefaciens. Barnase was modified by inserting an additional insert at a pivotal Ile-54 site, which could take crucial affect on protein structure and absolute activity. The lacZ' expressing cassette of pUC19 was replaced by the modified barnase under the NptIII promoter. This novel vector could exist in large quantities as pUC19 in E. coli hosts. For the direct cloning PCR fragments, the positive selective vector was prepared by linearizing pBN with EcoRV to cut off the additional insert. PCR fragments with different length were prepared to verify this vector by ligation with this vector. The results showed that this positive selective vector for PCR fragment cloning was higher efficient and more convenient in manipulation than previous positive vectors.
You
Leiming
L
Department of Bioengineering, Zhengzhou University, 100 Kexue Road, Zhengzhou, People's Republic of China.
Weng
Haibo
H
Chen
Zhankuan
Z
Wang
Aiping
A
Xu
Weihua
W
Wang
Minge
M
Dong
Ziming
Z
D001426:Bacterial Proteins; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D017433:Protein Structure, Secondary; D012260:Ribonucleases / Q000235:genetics*; D012641:Selection, Genetic
D001426:Bacterial Proteins; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease