[Construction of Corynebacterium glutamicum/E. coli shuttle promoter-probe vector]

Based on the replication origins of the C. glutamicum pXZ10145 and the Escherichia coli ColE1 plasmid, a novel Corynebacterium glutamicum/Escherichia coli shuttle vector pAK6 was constructed. This vector was able to replicate in C. glutamicum and E. coli. Plasmid pAK6 carried multiple cloning site useful for gene cloning, kanamysin- and ampicillin-resistance-encoding gene. Furtherly based on the shuttle vector pAK6, a promoter-probe vector was developed for the isolation of promoter elements from C. glutamicum . This vector carried the promoterless chloramphenicol acetyltranstersae (CAT) gene as a reporter downstream from useful cloning site. For testing this promoter-probe vector, C. glutamicum genomic DNA was digested to completion with Sau3AI and the fragments shot-gun cloned into its unique Bgl II. Two fragments exhibiting promoter activity were isolated. By measuring CAT activity, the strength of promoter fragments was assayed. After being sequenced, promoter sequences were predicted by using BDGP Neural Network Promoter Prediction V2.2 and the similarities to the regions of the consensus promoter sequence or the known promoters were confirmed.