| Project/Area Number |
15K14434
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| Research Category |
Grant-in-Aid for Challenging Exploratory Research
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| Allocation Type | Multi-year Fund |
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| Research Field |
System genome science
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| Research Institution | Tokyo Institute of Technology |
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Principal Investigator |
Iwasaki Hiroshi 東京工業大学, 科学技術創成研究院, 教授 (60232659)
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| Research Collaborator |
ITO Takehiko 東京工業大学, 生命理工学院, 教授 (90501106)
Palihati Maierdan 東京工業大学, 生命理工学院, 大学院生
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| Project Period (FY) |
2015-04-01 – 2017-03-31
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| Project Status |
Completed (Fiscal Year 2016)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000)
Fiscal Year 2016: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2015: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
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| Keywords | Cryptococcus酵母 / 形質転換 / 分子生物学 / 銅耐性 / 分子生物学ツール / モデル生物 / Cryptococcus / 酵母 / ゲノム編集 / ウラシル要求性 / 相同組換え / Cryptococcus 酵母 / 銅イオン / プラスミド |
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| Outline of Final Research Achievements |
Cryptococcus liquefaciens N6 is a unique strain of yeast that shows resistance to copper toxicity. To elucidate the mechanism underlying this phenomenon, we aimed to establish molecular biology tools in C. liquefaciens. For this, we first isolated ura- mutants by using 5-FOA as a positive selection marker. Two ura- mutants were found to have the same nonsense mutation in the URA5 gene by whole genome sequencing. Next, we established conditions for genetic manipulation of one ura5 mutant by electroporation, resulting in ura--to-ura+ transformation. We then attempted gene disruption of URA5 through a double crossover event with a targeting vector containing the NAT resistance gene. Although we obtained a considerable number of NAT resistant transformants, the NAT resistance gene was found to have integrated in an ectopic location in all cases. We speculate that gene disruption by this approach was unsuccessful due to intrinsically inefficient homologous recombination in C. liquefaciens.
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