| Project/Area Number |
01550683
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
高分子物性
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| Research Institution | Hokkaido University |
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Principal Investigator |
TAKAI Mitsuo Hokkaido University, Fac. of Engn., Assoc. Prof., 工学部, 助教授 (50002019)
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| Co-Investigator(Kenkyū-buntansha) |
FUJIWARA Masashi Hokkaido University, Fac. of Engn., Assistant, 工学部, 助手 (30229075)
SHIMIZU Yuichi Hokkaido University, Fac. of Eugn., Assistant, 工学部, 助手 (80142694)
HAYASHI Jisuke Hokkaido University, Fac. of Engn., Professor, 工学部, 教授 (10001182)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1990: ¥200,000 (Direct Cost: ¥200,000)
Fiscal Year 1989: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Acetobacter xylinum / Bacterial Cellulose / Plasmid / Shuttle Vector / Transformation / Shotgun Cloning / Restriction Mapping / B-isopropylmalate dehydrogenase gene / ロイシン合成遺伝子 / ビオチン / サザンハイブリダイゼ-ション / キメラプラスミド / シャトルベクタ- / 形質転換 |
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| Research Abstract |
Cellulose - producing bacterium Acetobacter aceti subsp. xylinum IFO 3288 was found to harbor seven plasmids named pFF1-7. The pFF plasmids had molecular weights of 1.4 - 9.4 kb and they were characterized with restriction endonucleases. Shuttle vector pUF106 was constructed by ligation of pFF6 to Escherichia coli plasmid pUC18. It had unique restriction sites suitable for insertion of a foreign DNA fragment and conferred ampicillin resistance to a host. This vector transformed cellulose-producing Acetobacter xylinum ATCC 10245 as well as E. coli JM109. Thus shuttle vector pUF106 is available as a practical cloning vector for cellulose- producing bacteria.
As a model experiment, the beta -isopropylmalate dehydrogenase (beta -IPMDH) gene, which concerned with Leucine synthesis, of cellulose-producing A. xylinum ATCC 10245 were cloned by shotgun cloning method using leuB-mutant host E. coli C600 and plasmid vector pUC18. Plasmid vector pUL1, carrying a 6.5 kilobase pairs (kb) BamHI-fragment, conferred the beta -IPMDH activity to the wild type of E. coli C600. Restriction mapping and deletion analysis indicated that the beta -IPMDH gene was located on a 1.15kb HindIII-fragment of plasmid pULH1. The cloned beta -IPMDH gene was identified with the original genes of A. Xylinum ATCC 10245 by Southern hybridization method using a biotin-labeled probe.
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