title: Improved plasmid-based system for fully regulated off-to-on gene expression in Escherichia coli: application to production of toxic proteins.
abstract: In previous work transduction of Escherichia coli with phage λ particles carrying packaged plasmids was shown to provide complete off-to-on expression of plasmid-borne genes (Cronan, J.E., 2003. J. Bacteriol. 185, 6522-6529). The plasmids used contained the phage λcos site (and hence are cosmids) and were very efficiently packaged into λ phage particles in vivo upon induction of λ lysogens having an inactivated cos site. However, a shortcoming was that the stocks contained a variable fraction of infectious λ phage which arose by recombinational repair of the inactive cos site. I report that the construction of E. coli strains that eliminate the background of infectious phage and show that the system can be utilized to express proteins by the phage T7 RNA polymerase/pET vector system.
authors.affiliation: Department of Microbiology, University of Illinois, Urbana, IL 61801, USA. j-cronan@life.uiuc.edu
authors.forename: John E
authors.initials: JE
authors.lastname: Cronan
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics / Q000378:metabolism; D002701:Chloramphenicol; D003360:Cosmids / Q000235:genetics / Q000378:metabolism; D003470:Culture Media / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D008242:Lysogeny; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic; D039002:Prophages / Q000235:genetics*; D013696:Temperature; D013752:Tetracycline; D014161:Transduction, Genetic; D014170:Transformation, Genetic
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003470:Culture Media; D002701:Chloramphenicol; D012321:DNA-Directed RNA Polymerases; D013752:Tetracycline