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 promoter-repressor systems was constructed using the tac-lacI and lambda pL-cI promoter-operator-repressor systems. In particular, transcription of the cat gene and the fused cI gene is regulated by the tac promoter, while transcription of the lacI gene is controlled by the lambda pL promoter. In order to compare CAT production from this new system with a currently employed transcription control configuration, a control expression vector utilizing the constitutive repressor synthesis configuration was also constructed. In this construct, cat is under the control of the tac promoter, and lac repressor is provided from a single copy of the lacIq allele included in the plasmid. Induction results using different copy number vectors indicate that induced cat expression levels are at least twofold higher using the cross-regulation system which has very low basal expression. These results match well with previous mathematical modeling predictions indicating excellent control of basal expression and also higher cloned-gene expression post-induction over a broad range of copy numbers for a cross-regulation control configuration. Induction of the cross-regulation system both up-regulated the activation pathway and down-regulated the inhibition pathway, shifting the system steady-state from lac repressor expression to cat and cI expression. The control strategy presented here should be equally applicable to regulate transcription in diverse hosts.
Chen
W
W
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125.
Kallio
P T
PT
Bailey
J E
JE
D010582:Bacteriophage lambda; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000032:analysis; D015536:Down-Regulation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D007763:Lac Operon; D008969:Molecular Sequence Data; D009876:Operon; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012097:Repressor Proteins / Q000096:biosynthesis / Q000235:genetics; D015854:Up-Regulation
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015500:Chloramphenicol O-Acetyltransferase