| Project/Area Number |
16360408
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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 |
Biofunction/Bioprocess
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| Research Institution | Tokyo Institute of Technology |
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Principal Investigator |
TANJI Yasunori Tokyo Inst. Of Technol., Dept.Bioeng., Associate Prof., 大学院生命理工学研究科, 助教授 (00282848)
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| Co-Investigator(Kenkyū-buntansha) |
MIYANAGA Kazuhiko Tokyo Inst. Of Technol., Dept.Bioeng., Assistant Prof., 大学院生命理工学研究科, 助手 (40323810)
海野 肇 東京工業大学, 大学院・生命理工学研究科, 教授 (10087471)
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| Project Period (FY) |
2004 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥12,500,000 (Direct Cost: ¥12,500,000)
Fiscal Year 2006: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2005: ¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2004: ¥6,500,000 (Direct Cost: ¥6,500,000)
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| Keywords | green chemistry / bacteriophage / surface engineering / Escherichia coli / 緑色蛍光タンパク質 / キトサナーゼ |
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| Research Abstract |
We designed a bacteriophage T2 system to display proteins fused at the N terminus of the head protein small outer capsid (SOC) of T2 phage. To facilitate selection of chimeric phage, a T2 phage encoding the β-galactosidase gene (βgal) upstream of the soc gene was constructed. The phage, named T2βGal, produces blue plaques on agar plates containing XGal. Subsequently, a plasmid encoding the target protein upstream of soc was constructed and used to transform E.coli B^E cells. Transformed cells were infected with T2βGal and homologous recombination between phage DNA and the plasmid resulted in a chimeric phage which produced transparent plaques due to the excision of the βgal gene. Chitosanase of Bacillus sp. strain K17 (ChoK), consisting of 453 amino acids, was used as a model target protein. Recombinant T2 phage which produced ChoK was named T2ChoK. T2ChoK was produced from T2βGal at a recombination frequency of about 0.1 %. On the other hand, the value for T2IβGal produced from wild type T2 was 0.001 %. This new system enables us to select recombinant phage very quickly and accurately. The number of molecules of ChoK was calculated at 14.7 per single phage. Latent period, and burst size were estimated for chimeric phages.
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