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 tac ) and operator ( O tac ) 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 β-galactosidase (βGal) were observed which were independent of the presence of pFSP6. This basal level of βGal 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. βGal activity increased about 18–32-fold when the expression of rpoSP6 was induced, as compared with the βGal 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.