Akademik Veri Yönetim Sistemi
EUROPEAN BIOTECHNOLOGY CONGRESS 2023, Ljubljana, Slovenya, 4 - 06 Ekim 2023, sa.86, ss.33
This study aims to determine the abundance and stability of three different transcripts (ORF-1, ORF-3, and ORF-5) encoded by the plasmid pAnox1, isolated from Anoxybacillus gonensis 05S15. Actinomycin-D, a known transcription inhibitor, was used to inhibit transcription. Initially, the optimal Actinomy-cin D concentration was determined through incubation of the plasmid-containing isolate at 55°C in 2 ml of Degryse medium overnight, followed by application of various Actinomycin-D concentrations (ranging from 1 to 50 mg/ml) to the cells. Later, the pAnox1-carrying cells were recultured, and the next day, subcultured into fresh Degryse medium at a 1:100 ratio. Actinomycin-D at 10 μg/ml was added during the logarithmic growth phase, and incubation continued at 55°C. At 0, 15, 25, and 30 minutes of incubation, 2 ml of cell culture was centrifuged to collect cell pellets. RNA isolation was performed from the pellets obtained at each stage. Equal amounts of RNA were used to synthesize a total of 12 cDNA samples for the three transcripts, which were subsequently analyzed using qPCR. After confirming with melting curve analysis, the results indicated that the transcripts were stable up to 25 minutes, after which degradation occurred. The half-life values were calculated as 29 minutes for ORF-1, 13.64 minutes for ORF-3, and 14.87 minutes for ORF-5.