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Characterization of new B. thuringiensis plasmids and construction of E.coli-Bt shutter vectors using a modified plasmid capture system
- 언어ENG
- URLhttps://db.koreascholar.com/Article/Detail/290422
Bacillus thuringiensis 1-3 (Bt 1-3), belonging to subsp. aizawai (H7), showed different characteristics in plasmid profiles from type strain and had cry2A gene in addition to cry1Aa, cry1Ab, cry1C and cry1D. To clone its plasmids and construct E.coli-Bt shuttle vector, we constructed the plasmid capture system (PCS) by inserting attB sites including lacZ between transposable elements (designated as pPCS-Troy). Through in vitro transposition with total plasmids DNA of Bt 1-3, 53 clones were acquired and their sizes were approximately 10 kb. Based on the sequence analysis, they were classified in four groups showing similarities with four known Bt plasmids, pGI3, pBMB175, pGI1 and pGI2, respectively. One of pGI3-like clones, named as pBt1-3, was fully sequenced and its putative open reading frames (ORFs), Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified. The structure of pBt1-3 showed high similarity with pGI3 which is one of rolling-circle replication (RCR) group VI family. As a donor for construction of shuttle vector, pDonr-attPEm vector harboring erythromycin resistant gene between attP sites was constructed. Through BP recombination with pPCS-Troy-cloned Bt plasmids and pDonr-attPEm, erythromycin resistant gene was transposed to Bt plasmids. This scheme proposes that in vitro transposition using pPCS-Troy and BP recombination using pDonr-attPEm can easily clone Bt plasmids and construct novel shuttle vectors.
