Maximizing gene expression on a plasmid using recombination in vitro

Abstract

Recombination in vitro has been used to place one or more copies of a strong promoter, the lac promoter, at varying distances from the cl (repressor) gene of bacteriophage lambda on the E. coli plasmid pMB9. In all constructions, lambda repressor synthesis is driven wholly or predominantly by the inserted lac promoter. One of our fusions directs the synthesis of very high levels of lambda repressor. In this case, the fused DNA encodes a ribosome binding site which is a "hybrid" of lambda and lac sequences. In principle, this method of construction should elicit high levels of expression in E. coli of any gene, whatever its source. We also described strains with different sequence arrangements that, for reasons not completely understood, produce less repressor.