DESIGN OF EXPRESSION VECTORS BASED ON pCR2 REPLICON OF Corynebacterium renale

A 3.2 kb plasmid pCR2 was isolated from Corynebcterium renale which harbours four cryptic plasmids. This plasmid was used for the construction of a range of cloning and expression vectors for E.co/i. This previously uncharacterized plasmid was first sequenced by cloning its fragments into pUC19. The complete nucleotide sequence thus obtained was annotated and submitted to GENEBANK (Accession No. EF488047). A complete restriction map was generated from the sequence obtained to identify dispensable restriction sites which are suitable for cloning. As a first step in the construction of vectors based on this replicon, antibiotic resistance cassettes viz ampicillin and chloramphenicol were introduced into the pCR2 backbone. These constructs (pCR2amp and pCR2cat) were used to transform E.coli DH5c cells. These plasmids were tested for stability in E.coli in the absence of selection pressure and a high level stability for a large number of generations was observed with these plasmids. Since this plasmid was quite different from known E.coli plasmids, it was expected that pCR2 would be compatible with known E.coli replicons. Compatibility studies were therefore carried out with two commonly used E.coli plasmids viz pUC19 (pMB1 ori) and pACYC184 (p15A ori). Both the plasmids (pCR2cat and pUC19 or pCR2amp and pACYC184) were stably maintained for 60 generations in shake flask cultures in the absence of selection pressure demonstrating sustained co-existence. Once the stability and compatibility of pCR2 had been demonstrated it was used to construct a range of expression vectors with different promoters and reporter