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젖산균을 이용한 대사공학적 도구의 개발
- Author(s)
- 채한승
- Advisor
- 이평천
- Department
- 일반대학원 분자과학기술학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2011-08
- Language
- eng
- Alternative Abstract
- As a part of a study identifying plasmids in lactic acid bacteria, I isolated and characterized two novel cryptic plasmids. The smaller of the two cryptic plasmids, pMBLR00, was isolated from Leuconostoc mesenteroides subsp. mesenteroides KCTC 3733. Complete sequencing of pMBLR00 revealed a 3370 bp element with G+C content of 36% harboring three open reading frames (ORFs A, B and C). The ORF A gene product has 98% amino acid identity with the Rep protein of a rolling-circle replication (RCR) plasmid pYSI8, isolated from Lactobacillus sakei YSI8. Southern hybridization and mung bean nuclease treatment demonstrated that pMBLR00 contains a single-stranded intermediate, confirming that pMBLR00 replicates via RCR mechanism. The ORF B gene product is homologous to mobilization proteins encoded by plasmids isolated from Leuconostoc and Lactobacillus species. An E. coli- Leuconostoc shuttle vector, pMBLR02, was constructed based on pMBLR00. The shuttle vector pMBLR02 was stable in Leuconostoc citreum 95. The larger plasmid, designated pMBLT00, was isolated from Leuconostoc mesenteroides subsp. KCTC 13302. This plasmid has a molecular size of 20721 bp and a G+C content of 38%. The sequence suggested that pMBLT00 contains of two major ORFs encoding Mob and Rep proteins. Two ORFs showed high homology with a replication initiation protein of the theta-type plasmid pLCK3, isolated from Leuconostoc citreum KM20. The putative Mob-Rep proteins have 84% and 95% amino acid identity with the plasmid pLCK3. Using Southern hybridization analysis, no single-stranded intermediates was detected, which indicates that pMBLT00 replicated via the theta mechanism. A new shuttle vector, pMBLT02, was constructed based on pMBLT00 minimal replicon. Furthermore, the pMBLT02 was stably maintained in Leuconostoc citreum 95 (for 100 generations) in the absence of erythromycin. In conclusion, this study revealed that we report on the occurrence of two differently replication (rolling circle replication and theta replication) plasmid in Leuconostoc strains (KCTC 3733, 13302), the cloning, complete sequencing and functional characterization of these elements, and its use as a cloning vector. In addition, we compared the stability of these two different replicating modes. The result indicated that two plasmids have the potential to be developed into valuable tools with metabolic engineering application.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Molecular Science and Technology > 3. Theses(Master)
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