Cloning and overexpression of the colicin E1 immunity gene

A DNA fragment containing only the putative immunity gene-coding sequence was cloned under the control of the trp and lambda PL promoters, generating pRKA11 and pIPL, respectively. Escherichia coli hosts containing either construction were immune to colicin E1. Cells harboring both pIPL and pNT204, which encodes a temperature-sensitive cI repressor, were sensitive to colicin E1 at 30 degrees C, but became immune after 0.5 h of incubation at 42 degrees C. In addition, pRKA11 directed the synthesis of a 14.5-kDA protein in maxicells, identical to that found with the wild-type immunity gene. This evidence identifies unequivocally the coding sequence of the immunity gene as that extending from bases 1214 to 1552 [OKA, A., et al., Mol. Gen. Genet. 172, 151-159 (1979)]. The entire immunity gene operon was also cloned under the control of the tac promoter, generating pTCU2, which, upon induction with isopropyl beta-D-thiogalactopyranoside, produced the imm gene product in amounts sufficient to be visualized by autoradiography.