A Standardized Vector System for Manipulation and Enhanced Expression of Genes inEscherichia coli

Abstract

Different families of cloning and expression vectors were engineered on a standard plasmid. They contain several regulatory signals for transcription and/or translation initiation and termination. The plasmids in each series differ only in the number, type, and order of unique restriction cleavage sites clustered in front of a transcription terminator. The pLK30 plasmids are general cloning vectors and the corresponding pLK50 plasmids carry the λp_L promoter. The pLK60 vectors carry the λp_R promoter and translation initiation signals of the era gene containing the Shine–Dalgarno sequence and initiation codon. The pLK70 series is similar to pLK60 except that additional 5′-translated cro sequences are included. The pLK80 plasmids have a lacZ gene fragment suitable for the construction of hybrid genes. The presence of translational stop signals in the pLK90 series facilitates the manipulation of genes truncated at the 3′ end. This standardized pLK vector system offers great versatility in gene manipulation and in optimization of gene expression under the control of strong regulatable promoters. Measurement of expression levels under repressed conditions permits the identification of optimal promoter-gene configurations in constructions directing high-level expression.