『徳島大学 教育・研究者情報データベース (EDB)』---[学外] /
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種別 (必須): 学術論文 (審査論文) [継承]
言語 (必須): 英語 [継承]
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著者 (必須): 1. (英) Ichimura Minoru (日) (読)
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2.中山 治之
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3. (英) Wakimoto Shin (日) (読)
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4. (英) Morita Hidetoshi (日) (読)
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5. (英) Hayashi Tetsuya (日) (読)
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6.桑原 知巳
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題名 (必須): (英) Efficient electrotransformation of Bacteroides fragilis.  (日)    [継承]
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要約 (任意): (英) This study describes refined electroporation parameters for efficient transformation of Bacteroides fragilis by plasmids prepared from laboratory strains of Escherichia coli. Development of the method used included determination of the optimal growth conditions for competent cell preparation, selectable antimicrobial resistance markers, electric field strength, and postpulse incubation time. Of the four E. coli-Bacteroides shuttle plasmids tested (pVAL-1, pVAL-2, pNLY1, and pLYL05), pLYL05 containing the cefoxitin resistance marker was found to be the most suitable for B. fragilis transformation, and it generated 2- to 900-fold more transformants (about 10(4) transformants per microg pLYL05 DNA) than the other plasmids. For the 72-h cultivation period tested, B. fragilis cells harvested at 48 h yielded the highest numbers of transformants. The transformation efficiency of pLYL05 increased linearly with the electric field strength over a range from 5.0 to 12.5 kV/cm. At least 3 h of postpulse incubation was required to maximize the transformation efficiency. For deletion of B. fragilis genes by homologous recombination, competent cells grown to early exponential phase and 12 h of postpulse incubation were required for efficient integration of the pLYL05-based suicide vector into the target site. The expected integration was obtained in B. fragilis strain NCTC9343 only when a homologously prepared (i.e., in vivo methylated) suicide vector was used. Spontaneous resolution of the diploid successfully deleted the expected genetic region. Our simple and efficient plasmid transfer method enabled disruption of a B. fragilis gene using in vivo-methylated targeted vectors. Our optimized electroporation parameters provide a useful tool for genetic manipulation of Bacteroides species.  (日)    [継承]
キーワード (推奨): 1. (英) Bacteroides fragilis (日) (読) [継承]
2. (英) Electroporation (日) (読) [継承]
3.大腸菌 (Escherichia coli) [継承]
4. (英) Plasmids (日) (読) [継承]
5. (英) Sequence Deletion (日) (読) [継承]
6. (英) Time Factors (日) (読) [継承]
7. (英) Transformation, Genetic (日) (読) [継承]
発行所 (推奨):
誌名 (必須): Applied and Environmental Microbiology ([アメリカ微生物学会])
(pISSN: 0099-2240, eISSN: 1098-5336)

ISSN (任意): 1098-5336
ISSN: 0099-2240 (pISSN: 0099-2240, eISSN: 1098-5336)
Title: Applied and environmental microbiology
Title(ISO): Appl Environ Microbiol
Publisher: American Society for Microbiology
 (NLM Catalog  (Scopus  (CrossRef (Scopus information is found. [need login])
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(必須): 76 [継承]
(必須): 10 [継承]
(必須): 3325 3332 [継承]
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年月日 (必須): 西暦 2010年 5月 初日 (平成 22年 5月 初日) [継承]
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DOI (任意): 10.1128/AEM.02420-09    (→Scopusで検索) [継承]
PMID (任意): 20348295    (→Scopusで検索) [継承]
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備考 (任意): 1.(英) Article.Affiliation: Department of Molecular Bacteriology, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan.  (日)    [継承]
2.(英) Article.PublicationTypeList.PublicationType: Journal Article  (日)    [継承]
3.(英) Article.PublicationTypeList.PublicationType: Research Support, Non-U.S. Gov't  (日)    [継承]
4.(英) OtherID: PMC2869128  (日)    [継承]

標準的な表示

和文冊子 ● Minoru Ichimura, Haruyuki Nakayama, Shin Wakimoto, Hidetoshi Morita, Tetsuya Hayashi and Tomomi Kuwahara : Efficient electrotransformation of Bacteroides fragilis., Applied and Environmental Microbiology, Vol.76, No.10, 3325-3332, 2010.
欧文冊子 ● Minoru Ichimura, Haruyuki Nakayama, Shin Wakimoto, Hidetoshi Morita, Tetsuya Hayashi and Tomomi Kuwahara : Efficient electrotransformation of Bacteroides fragilis., Applied and Environmental Microbiology, Vol.76, No.10, 3325-3332, 2010.

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