Functional Analysis Of A Novel, Cryptic, Small And Multicopy Plasmid (pHIG22) From Thermus Scotoductus K6

In this study, a small, novel, cryptic, multicopy and double strand plasmid (pHIG22) which has 2222 bp in long and with a total G + C content of 62,78 from Thermus scotoductus K6 was characterized and functionally analyzed. A 2012 bp region among 387 and 614 bp of pHIG22 was determined as minimal replicon of plasmid. Replication mode of pHIG22 was identified as 'Teta' and rep origin of it was determined as a region among 46 and 239 (BamHI recognition site is accept as '0' point) bp by TEM analysis. Two transcripts were detected from the both strands of pHIG22. According to RACE and LACE analysis results, Transcript 1 (329-1950) and Transcript 2 (2103-329) were determined 1622 bp in long and 1712 bp in long, respectively. Analysis of SDS-PAGE, Zimogram, 2D gel analysis and MALDI-TOF/TOF analysis show that transcripts do not encode any proteins, and these studies were performed to understand that transcripts whether encode or not encode proteins. The regulator regions of transcripts were investigated and promotor sequences were detected on both strands. The copy number of pHIG22, which has no ability to replicate in E.coli strains, in T. scotoductus K6 was calculated as 148 by using Real-time qPCR technique. pUCHIGK shuttle cloning vector and pHIGKE2 shuttle expression vector, which can replicate in E.coli-Thermus strains, were constructed by using pHIG22 minimal replicon and various DNA fragments.