A new T7 RNA polymerase-driven expression system induced via thermoamplification of a recombinant plasmid carrying a T7 promoter-Escherichia coli lac operator
| Autor: | Nikolay A. Tsyba, Lebedeva Mi, Sergey V. Mashko, Ekaterina V. Rogozhkina |
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| Rok vydání: | 1994 |
| Předmět: |
Chloramphenicol O-Acetyltransferase
Isopropyl Thiogalactoside Operator Regions Genetic Transcription Genetic lac operon Repressor Lac repressor Biology Viral Proteins Transcription (biology) Bacteriophage T7 Gene expression Escherichia coli Genetics medicine T7 RNA polymerase Cloning Molecular Promoter Regions Genetic Gene Gene Amplification Temperature RNA DNA-Directed RNA Polymerases General Medicine Molecular biology Lac Operon Plasmids medicine.drug |
| Zdroj: | Gene. 142:61-66 |
| ISSN: | 0378-1119 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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