CONSTRUCTION OF LACITS AND LACI(Q)TS 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 contro l element, we have (i) mapped the mutation to the inducer-binding doma in 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, le ading to a Gly(187)-->Ser substitution, (iii) removed extraneous lacZo p 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 lacI(q ) promoters. The LacIts repressor is insensitive to IPTG. The repressi on of lacZop by LacIts is very efficient at 30 degrees C and total ind uction was achieved at 42 degrees C, providing that the LacIts concent rations are not excessive and that repressor-to-operator ratios are no t too high.