Analysis of replicative properties of pAnox1 plasmid by in silico and in vitro experiments

In this study, the replicative properties of transcripts encoded by pAnox1, plasmid harbored by Anoxybacillus gonensis 05S15 were investigated by in silico and in vitro analyses.In silico and in vitro analyses were performed for three transcripts (ORF-1, ORF-3, ORF-5) known to be encoded by the pAnox1 plasmid. In the studies conducted to determine whether the transcripts were translated into proteins, it was found that the ORF-1 transcript encoded a protein based on plasmid curing, SDS-PAGE, and silver staining analyses, but ORF-3 and ORF-5 transcripts were not translated into proteins. It was revealed that the ORF-1 transcript encoded 903 bp, ORF-3 transcript 499 bp, and ORF-5 transcript 211 bp. Using quantitative real-time PCR, the intracellular copy number of pAnox1 was determined to be approximately 127±2. The protein responsible for the replication of pAnox1 was cloned into the pET28a(+) vector, overexpressed in E. coli, and purified. Bioinformatic analyses and docking simulations identified the regulatory regions of the plasmid and the binding properties of the Rep protein.