Using chromosomal lacIQ1 to control expression of genes on high-copy-number plasmids in Escherichia coli.

Transcription of the lac and the hybrid tac promoters is repressed by the lac repressor and induced by the non-metabolizable substrate IPTG. The degree of repression depends upon the ratio of LacI molecules in a cell to the DNA operator sites. In the absence of an inducer, repression of Ptac on a high-copy-number (hcn) plasmid was equivalent in strains containing lacIQ1 on the chromosome, or lacI+ on the plasmid, but not from strains with lacI+ or lacIQ only on the chromosome. Induction of Ptac on hcn plasmids in strains in which expression was controlled by lacIQ1 occurred at very low inducer concentrations (3‐10 mM IPTG) and reached levels significantly higher than in strains with lacI+ on the plasmid. Greater than 300-fold induction of a bLacZ fusion was observed, and >600-fold induction was estimated from recombinant hemoglobin synthesis. Transcription from PlacIQ1 initiated in the same point as PlacI+, but was 170-fold stronger, consistent with the lac repressor levels required to control LacI-regulated genes on hcn plasmids. The DNA sequence upstream of lacI was used to develop a simple PCR test to identify lacIQ1 by a characteristic 15-bp deletion. This deletion created a consensus ’35 hexamer, responsible for the increased lacI transcription, and was easily detectable in a variety of strains. Using lacIQ1 hosts eliminates the requirement to maintain lacI on the plasmid to regulate gene expression on hcn expression plasmids. © 1998 Elsevier Science B.V. All rights reserved.