| Project/Area Number |
16380066
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied microbiology
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| Research Institution | Mitsubishi Kagaku Institute of Life Sciences |
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Principal Investigator |
ITAYA Mitsuhiro Mitsubishi Kagaku Institute of Life Sciences, Research Department, Senior Researcher, 研究部門, 主任研究員 (60374013)
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| Co-Investigator(Kenkyū-buntansha) |
FUJITA Kyoko Mitsubishi Kagaku Institute of Life Sciences, Research Department, Technical Assistant, 研究部門, 技術員
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥9,500,000 (Direct Cost: ¥9,500,000)
Fiscal Year 2005: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2004: ¥5,500,000 (Direct Cost: ¥5,500,000)
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| Keywords | genome engineering / Bacillus subtilis transformation / conjugational transfer / antibiotic resistance marker / Bacillus natto / 枯草菌ゲノム / プラスミド / 液体培養 / 固体培地 / typeIV secretion system / 薬剤選択 / 塩基配列 / 足場配列 / リピート配列 |
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| Research Abstract |
pLS20-mediated conjugational transfer between Bacillus subtilis was investigated. The first achievement was that construction of a pLS20 plasmid marked by an antibiotic resistance gene, cat. This was based on the determined sequence of the 65-kb pLS20 plasmid. Next, the assay method for conjugation was established not on conventional solid media but in liquid culture. By this, detailed conjugational kinetics was possible and revealed that pLS20 transmission occurred at a limited cellular growth stage of both donor and recipient. This observation seems critical to study the mechanism underlying the typeIV secretion pathways deduced from the encoded genes by pLS20. The nucleotide sequence of other small pLS30 (6,610 bp) exhibited the gene mob and its recognition sequence oriT that features as mobile between Bacillus species on conjugational transfer. The plasmid pLS3001, a chimera with pBR322-based plasmid was prepared in Escherichia coli. Apparent mobilizing activity of this amall plasmid was found in the pLS20- mediated conjugational transfer system. The rep gene and associated palT1-like sequence common in the pLS30 indicated that pLS30 belongs to a typical rolling circle replicating (RCR) type plasmid. Taken together with the significant stability of pLS30 in its original strain, application of mobile plasmid would offer quick propagation of cloned genes to Bacillus species. It should be emphasized that mutation of the recipient recA did not significantly interfere with the pLS20 mediated conjugational transfer process. This finding will open an effective way to deliver giant DNAs to the B.subtilis recA mutant as is normal in the case of E.coli gene cloning.
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