title: A tightly regulated expression system in Escherichia coli with SP6 RNA polymerase.
abstract: A tightly regulated gene-expression system was developed using SP6 RNA polymerase (RpoSP6). The RpoSp6-encoding gene (rpoSP6) was inserted into a mini-F plasmid (mini-F) and expression was controlled by the lactose promoter (P(lac)) and operator (O(lac)) on the plasmid. Therefore, a controlled expression system for the target genes can easily be constructed in various host strains by co-transformation of the system plasmid pFSP6 with other vector plasmids containing the genes linked to the SP6 promoters (P(SP6)). Using the lac gene linked to P(SP6) as a reporter, we evaluated the regulation of expression in this system in various host strains. Low-level expression of lac was detected in Escherichia coli harboring this expression system when RpoSP6 was uninduced, although very low activities of beta-galactosidase (beta-Gal) were observed which were independent of the presence of pFSP6. This basal level of beta-Gal activity was possibly derived, because the P(SP6) element has very weak activity for E. coli RNA polymerase (Rpo). These results showed that RpoSP6 seemed to be produced at very low levels in uninduced cells. Beta-GAl activity increased about 18-32-fold when the expression of rpoSP6 was induced, as compared with the beta-Gal activity when uninduced. The tight regulation of this system is superior to that of other known systems and it has a considerable advantage for gene expression in E. coli.
authors.affiliation: Genetic Engineering Section II, Biotechmology Research Laboratories, Takara Shuzo Co., Ltd., Shiga, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Sagawa
authors.forename: A
authors.initials: A
authors.lastname: Ohshima
authors.forename: I
authors.initials: I
authors.lastname: Kato
mesh_terms: D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D015151:Immunoblotting; D007763:Lac Operon / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: C070852:RNA polymerase SP6; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase