[Construction of promoter probe vector for a cold-adapted bacterium, Acinetobacter sp. DWC6]

In order to build a protein expression system in a cold-adapted bacterium Acinetobacter sp. DWC6, a promoter probe vector was constructed based on the plasmid pBR322. A fragment containing the promoter of the beta-lactamase gene (the ampicillin resistance gene) in pBR322 was eliminated and replaced by a fragment comprizing a kanamycin resistance gene amplified from pJRD215. DNA fragment harboring in the Acinetobacter species specific ori was also inserted into the plasmid pBR322 to construct a promoter probe vector named pBAP1, which could replicate both in E. coli and in Acinetobacter sp. DWC6. The promoter selection library was constructed by randomly inserting genomic DNA fragment of Acinetobacter sp. DWC6 at upstream of reported gene, and target promoters were screened from genomic library on ampicillin selection plates. The function of pBAP1 and isolated promoters were determined by detection of the ampicillin sensitivity and the expression level of beta-lactamase in the host cell.