A new T7 RNA polymerase-driven expression system induced via thermoamplification of a recombinant plasmid carrying a T7 promoter-Escherichia coli lac operator.
A new temperature-regulated T7 RNA polymerase-driven transcription system has been developed. This system is based on a hybrid regulatory region: the phage T7 late promoter (PT7) linked to the Escherichia coli lac operator (Olac) [Giordano et al., Gene 84 (1989) 209-219], which was located in an earlier obtained [Mashko et al., Gene 97 (1991) 259-266] temperature-controlled amplifiable plasmid, carrying cat under the control of PT7-Olac and, in addition, lambda major early promoter-operator regions and gene cIts857. Plasmids of the pT7-Olac-cat-tsr series were stably maintained at a low-copy-number when grown at low temperature (28 degrees C). In E. coli BL21(DE3), carrying the Plac-controllable T7 RNA polymerase-encoding gene, efficient repression of cat transcription was observed, that was provided by the LacI repressor and, probably, the thermolabile repressor CIts857. At low and moderate temperatures (28/37 degrees C), this 'cooperative' repression was so tight that cat expression was not observed in the cells carrying PT7-Olac on the plasmids, even after IPTG-inducible T7 RNA polymerase biosynthesis. As a result of the thermo-amplification of the recombinant plasmids and temperature-inactivation of CIts857, expression of the T7 RNA polymerase-encoding gene was derepressed due to the titration of LacI by the increasing copies of Olac which in turn, led to the highly efficient T7 RNA polymerase-driven accumulation of CAT in the cells.
Lebedeva
M I
MI
Institute of Genetics and Selection of Industrial Microorganisms, Moscow, Russia.
Rogozhkina
E V
EV
Tsyba
N A
NA
Mashko
S V
SV
D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005784:Gene Amplification / Q000187:drug effects; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007763:Lac Operon; D009875:Operator Regions, Genetic; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D013696:Temperature; D014158:Transcription, Genetic; D014764:Viral Proteins
D014764:Viral Proteins; D007544:Isopropyl Thiogalactoside; D015500:Chloramphenicol O-Acetyltransferase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases