CONSTRUCTION AND CHARACTERIZATION OF A NOVEL CROSS-REGULATION SYSTEM FOR REGULATING CLONED GENE-EXPRESSION IN ESCHERICHIA-COLI

A novel cross-regulation system employing two pairs of interacting pro moter-repressor systems was constructed using the tac-lacI and lambda p(L)-cI promoter-operator-repressor systems. In particular, transcript ion of the cat gene and the fused cI gene is regulated by the tac prom oter, while transcription of the lacI gene is controlled by the lambda P(L) promoter. In order to compare CAT production from this new syste m with a currently employed transcription control configuration, a con trol expression vector utilizing the constitutive repressor synthesis configuration was also constructed. In this construct, cat is under th e control of the tac promoter, and lac repressor is provided from a si ngle copy of the lacI(q) allele included in the plasmid. Induction res ults using different copy number vectors indicate that induced cat exp ression levels are at least twofold higher using the cross-regulation system which has very low basal expression. These results match well w ith previous mathematical modeling predictions indicating excellent co ntrol of basal expression and also higher cloned-gene expression post- induction over a broad range of copy numbers for a cross-regulation co ntrol configuration. Induction of the cross-regulation system both up- regulated the activation pathway and down-regulated the inhibition pat hway, shifting the system steady-state from lac repressor expression t o cat and cI expression. The control strategy presented here should be equally applicable to regulate transcription in diverse hosts.