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.