Construction of lacIts and lacIqts expression plasmids and evaluation of the thermosensitive lac repressor.
To characterize a thermosensitive lacIts mutant isolated by Bukrinsky et al. [Gene 70 (1989) 415-417] and to adapt it as a convenient control element, we have (i) mapped the mutation to the inducer-binding domain of the LacIts repressor, (ii) determined that the mutation resulted in the loss of a HaeIII site in lacIts due to a G-->A transition, leading to a Gly187-->Ser substitution, (iii) removed extraneous lacZop DNA downstream of lacI, and (iv) cloned lacIts (in plasmids based on ori of either ColE1 or P15A) under control of the wild-type or lacIq promoters. The LacIts repressor is insensitive to IPTG. The repression of lacZop by LacIts is very efficient at 30 degrees C and total induction was achieved at 42 degrees C, providing that the LacIts concentrations are not excessive and that repressor-to-operator ratios are not too high.
Hasan
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N
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison 53706-1599, USA.
Szybalski
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W
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics*; D003001:Cloning, Molecular; D004790:Enzyme Induction / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015870:Gene Expression / Q000187:drug effects; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D056968:Lac Repressors; D007785:Lactose / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000235:genetics*; D015183:Restriction Mapping; D013696:Temperature; D001616:beta-Galactosidase / Q000096:biosynthesis
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D011994:Recombinant Proteins; D012097:Repressor Proteins; D007544:Isopropyl Thiogalactoside; D001616:beta-Galactosidase; D007785:Lactose