低温菌启动子探针质粒的构建

为了在宿主菌Acinetobacter sp.DWC6中构建低温菌蛋白表达载体,以pBR322质粒为基础,去除质粒上β-内酰胺酶基因的启动子片段,取而代之为来源于质粒pJRD215的卡那霉素抗性基因片段,并在pBR322中插入Acinetobacter菌属特异性ori的DNA片段,构建了能在Acinetobacter sp.DWC6和E.coli中正常复制的启动子探针质粒pBAP1。通过在质粒pBAP1中的β-内酰胺酶基因上游随机导入Acinetobacter sp.DWC6基因组片段,通过检测宿主细胞的氨苄青霉素抗性和β-内酰胺酶活性,来筛选强启动子片段,并分析了启动子探针质粒载体的功能及启动子的强度。

Construction of Promoter Probe Vector for a Coldadapted 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.