A family of Corynebacterium glutamicum/Escherichia coli shuttle vectors for cloning, controlled gene expression, and promoter probing

Abstract

A new family of vectors including cloning vectors (pEK0; pEC5), an expression vector (pEKEx1), and promoter probe vectors (pEKpllacZ; pEKplCm), has been constructed. All these shuttle vectors are based on the replication origins of the corynebacterial pBL1 and the Escherichia coli ColE1 plasmids, and thus are able to replicate in Corynebacterium glutamicum and E. coli. Plasmids pEK0 and pEC5 carry multiple restriction sites useful for gene cloning and the kanamycin- or chloramphenicol-resistance-encoding gene from Tn903 or from Tn9, respectively. In C. glutamicum, both vectors are compatible with vectors containing the corynebacterial pHM1519 replicon. Based on plasmid pEK0, the expression vector pEKEx1 was developed to allow for isopropyl-beta-D-thiogalactopyranoside-inducible expression of inserted genes in C. glutamicum and E. coli. Also based on pEK0, the promoter probe vectors pEKpllacZ and pEKplCm were constructed to carry the promoterless lacZ or cat reporter genes downstream from useful cloning sites, for assaying the transcriptional activity of cloned fragments.