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Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication
Authors:E Remaut  H Tsao  W Fiers
Institution:Laboratory of Molecular Biology, State University of Ghent, Ledeganckstraat, 35, 9000-Ghent Belgium Tel. (091) 22 78 21
Abstract:Improved expression vectors have been constructed which are derived from runaway-replication mutants of plasmid R1 and carry the strong leftward promoter (pL) of bacteriophage lambda. The activity of this promoter is controlled by a temperature-sensitive repressor, product of the phage gene cI cloned on a compatible plasmid. Heat induction leads to amplification of the plasmid copy number and at the same time turns on the promoter. At a short distance downstream from the promoter, unique EcoRI, BamHI, XbaI and HindIII sites are present. This system was used for high level expression of the T4 DNA-ligase gene; 3 h after induction the ligase amounted to about 20% of total cellular protein.
Keywords:Recombinant DNA  pKN402 derivatives  T4 DNA ligase production  resistance to ampicillin and carbenicillin  bp  base pairs  CAT  chloramphenicol acetyltransferase  Δ  deletion  EtBr  ethidium bromide  IPTG  resistance to kanamycin  LB  see MAT  & METH    section (a)  major leftward bacteriophage λ promoter  resistance to streptomycin  SDS  sodium dodecyl sulfate  TCA  trichloroacetic acid  [ ]  indicates plasmid-carrier state
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