Characterization of temperature inducible promoters from a novel rolling circle replicating plasmid of Enterococcus faecium DJ1

Limited studies have been performed on the characterization of small size plasmids of Enterococcus faecium with the intention of evaluating the strength of their promoters in Escherichia coli. The complete nucleotide sequence (3.825 Kb) and structural organization of E. faecium DJ1 cryptic plasmid pNJAKD is presented. Seven promoter sequences from the pNJAKD plasmid of E. faecium have been identified. The regions coding for the putative promoters were either amplified using PCR based techniques or chemically synthesized as oligonucleotides of different sizes. These were subsequently cloned in the pEGFP vector at the Pvu II site. The efficiency of putative promoter fragments were measured using the intensity of eGFP fluorescence in E. coli JM101, DH5α and BL21(DE3), among which AKD3 exhibited moderate to strongest promoter activity at temperatures of 30, 37, and 42°C.