Construction of plasmid cloning vehicles that promote gene expression from the bacteriophage lambda pL promoter.
Two multiple-copy, ColE1-type, plasmid cloning vehicles, pHUB2 and pHUB4, have been constructed that carry four different single restriction sites down-stream from the phage lambda promoter pL. The promoting activity of pL is switched off at low temperature in the presence of a cIts gene that specifies a temperature-sensitive repressor but could be activated by heat induction. cIts was located either on the host chromosome, or on a second plasmid pRK248 that is compatible with the cloning vehicle, or on the vehicle itself. Three different restriction fragments, each carrying the gene trpA of Salmonella typhimurium or Shigella dysenteriae, have been inserted into the EcoRI, BamHI and SalI sites, respectively, of these plasmids and pL dependent expression of the inserted gene in Escherichia coli was determined by measuring the enzymatic activity of the trpA gene product. Heat induction resulted in a level of expression of trpA corresponding to 1 to 6.6% of the total soluble cell protein as trpA protein. The level of trpA protein production depended on the particular insert and the plasmid used.
D001426:Bacterial Proteins / Q000235:genetics; D003090:Coliphages / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000235:genetics; D004274:DNA, Recombinant; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D009876:Operon; D010641:Phenotype; D010957:Plasmids; D011995:Recombination, Genetic; D012486:Salmonella typhimurium / Q000235:genetics; D012762:Shigella dysenteriae / Q000235:genetics; D013696:Temperature; D014158:Transcription, Genetic; D014364:Tryptophan / Q000235:genetics*; D014367:Tryptophan Synthase / Q000235:genetics
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D004279:DNA, Viral; D014364:Tryptophan; D004262:DNA Restriction Enzymes; D014367:Tryptophan Synthase