A novel plasmid (pAnox1) from Anoxybacillus sp. 05S15


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Ayyıldız H., Çubukcı G., Güler H. İ. , İnan Bektaş K. , Beldüz A. O.

Molbiyokon'22, 8th International Congress of the Molecular Biology Association of Turkey, İstanbul, Turkey, 9 - 12 June 2022, pp.1-2

  • Publication Type: Conference Paper / Summary Text
  • City: İstanbul
  • Country: Turkey
  • Page Numbers: pp.1-2

Abstract

Abstract
Beckground/aim: Thermophilic bacteria has been one of the most interesting research focuses in recent years. Although plasmids are common among all bacterial species, fewer plasmids have been isolated from thermophilic bacteria than from other bacterial species. In this study, a small, novel, double-stranded and cryptic plasmid was isolated from Anoxybacillus sp. 05S15 and was detected its stability within the host.
Materials and methods: Plasmid DNA was isolated from overnight culture of Anoxybacillus sp. 05S15 strain. To reveal the whole genome sequence of plasmid DNA, it was linearized with NdeI and HindIII restriction endonucleases, then cloned into the pUC18 vector. After sequencing, the whole genome sequence of the plasmid was obtained. According to the BLAST analysis results, it was determined that the plasmid DNA sequence did not show similarity to other plasmids. For the novel plasmid designated as pAnox1, analyzes were performed using bioinformatics software such as motif analyzer, ORF finder, rep protein determinator, promoter predictor, direct repeats-palindroms finder. In addition, intracellular stability test of pAnox1 plasmid was performed. For this purpose, overnight culture from Anoxybacillus sp. 05S15 isolate harbored pAnox1 was incubated in 50 ml of TSB medium. The culture was incubated for 16 hours at 55 °C, which is the optimum growth temperature of Anoxybacillus sp. 05S15 strain. Cells were continuously subcultured by this way and the 26th, 52nd, 78th, 104th and 139 th generations were spreaded on petri dishes. After incubation plasmid isolation performed from growing colonies to detect its stability.
Result: Sequence analysis results showed that Anoxybacillus sp. 05S15 strian harbored a novel plasmid (pAnox1) which has 1592 bp in long and with a total G + C content of 40,01. According to the results of bioinformatics analyzes performed with the NCBI OriFinder program, 5 different ORFs were detected in the pAnox1 genome. Moreover, three palindrome sequences and two iteron sequences were detected. The intracellular stability test, showed that the isolate harboured plasmids in the 26, 52, 78 and 104th generations, that is, in the 8th passage of the isolate. It was determined that the novel plasmid lost its stability after 130th generation.
Conclusion: The pAnox1 plasmid purified from Anoxybacillus sp. 05S15 was determined to be a novel plasmid with a cryptic, small genome, double-stranded, 1592 bp in length and total G+C content of 40,01%. According to the experimental and bioinformatic analysis results, pAnox1 can be developed for vector design and used in biotechnological studies.