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 (3-10microM IPTG) and reached levels significantly higher than in strains with lacI+ on the plasmid. Greater than 300-fold induction of a beta-LacZ 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.
Glascock
C B
CB
Somatogen, Inc., 2545 Central Avenue, Boulder, CO 80301, USA.
Weickert
M J
MJ
D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics*; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D007544:Isopropyl Thiogalactoside / Q000378:metabolism / Q000494:pharmacology; D056968:Lac Repressors; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D012097:Repressor Proteins / Q000032:analysis / Q000235:genetics*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D012097:Repressor Proteins; D007544:Isopropyl Thiogalactoside