Control of cloned gene expression by promoter inversion in vivo: construction of improved vectors with a multiple cloning site and the Ptac promoter.
We have constructed three gene-expression plasmids which contain (an) invertible promoter(s) and a multiple cloning site. We used either the plac promoter or the ptac-plac tandem promoters, the latter directing a more than fourfold increase in expression of the galK reporter gene in Escherichia coli host. All these plasmids were derived from the pNH7a expression plasmid of Podhajska et al. [Gene 40 (1985) 163-168]. Like pNH7a, these vectors have three novel properties: (i) in the 'OFF phase', the promoter is facing away from the gene to be expressed, (ii) the 'ON phase' is attained by the rapid and efficient inversion of the promoter mediated by the phage lambda Int product and the flanking attP and attB sites, which have a divergent orientation, and (iii) only a short heat pulse is required for the efficient inversion of the promoter and switching from the OFF to the ON phase. As for the pNH7 a vector, the present plasmids contain the nut-N transcriptional antitermination system, which permits efficient gene expression even if terminator(s) happen to be present between the promoter(s) and the expressed gene. The promoter inversion is rapid and over 95% efficient, as assayed by restriction analysis and galactokinase assay. Many genes could be conveniently cloned in the multiple cloning site, and then either kept totally silent or expressed in a rigidly controlled manner. Moreover, the pNH8, pNH16 and pNH18 plasmids, with already inverted promoters, could be used for expression of cloned genes, either in an unregulated manner or regulated by the lac repressor. They would be particularly useful for genes associated with terminators affecting their expression.
D001287:Attachment Sites, Microbiological; D010582:Bacteriophage lambda / Q000235:genetics; D007446:Chromosome Inversion; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D010957:Plasmids; D011401:Promoter Regions, Genetic