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Counterselection system employing mutated pheS for markerless genetic deletion in Bacteroides species
https://kagawa-u.repo.nii.ac.jp/records/402
https://kagawa-u.repo.nii.ac.jp/records/40248a947e7-7792-4dce-8090-91fde020ba4e
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本文 (5.1 MB)
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内容の要旨 (112.7 kB)
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審査の結果の要旨 (610.7 kB)
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| Item type | 学位論文 / Thesis or Dissertation(1) | |||||
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| 公開日 | 2019-07-30 | |||||
| タイトル | ||||||
| タイトル | Counterselection system employing mutated pheS for markerless genetic deletion in Bacteroides species | |||||
| 言語 | en | |||||
| 言語 | ||||||
| 言語 | eng | |||||
| 資源タイプ | ||||||
| 資源タイプ識別子 | http://purl.org/coar/resource_type/c_db06 | |||||
| 資源タイプ | doctoral thesis | |||||
| アクセス権 | ||||||
| アクセス権 | open access | |||||
| アクセス権URI | http://purl.org/coar/access_right/c_abf2 | |||||
| 著者 |
木納, 康博
× 木納, 康博 |
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| 抄録 | ||||||
| 内容記述タイプ | Abstract | |||||
| 内容記述 | Abstract Markerless gene deletion is necessary for multiple gene disruptions due to the limited number of antibiotic resistant markers for some bacteria. However, even in transformable strains, obtaining the expected mutation without a marker requires laborious screening of a large number of colonies. Previous studies had success in various bacteria with a counter-selection system where a conditional lethal gene was incorporated into the vector. We examined the efficacy of the mutated pheS gene (pheS*) as a counter-selective marker for gene deletion in Bacteroides. This mutation produces an amino acid substitution (A303G) in the alpha subunit of Bacteroides phenylalanyl tRNA synthetase, which in E. coli alters the specificity of the tRNA synthetase resulting in a conditional lethal mutation due to the incorporation of p-chloro-phenylalanine (p-Cl-Phe) into protein. B. fragilis YCH46 and B. thetaiotaomicron VPI-5482 transformed with a pheS*-harboring shuttle vector were clearly growth-inhibited in the presence of >5 mM p-Cl-Phe in liquid defined minimal media (DMM) and on DMM agar plates. A targeting plasmid was constructed to delete the genetic region for capsular polysaccharide PS2 in B. fragilis or PS1 in B. thetaiotaomicron. After counterselection, p-Cl-Phe-resistant colonies were generated at a frequency of 8.1 × 10−3 for B. fragilis and 1.7 × 10−3 for B. thetaiotaomicron. Of the p-Cl-Phe-resistant colonies, 4.2% and 72% harbored the correct genetic deletion for B. fragilis and B. thetaiotaomicron, respectively. These results indicate that mutated pheS is a useful counter-selective gene to construct markerless genetic deletions in Bacteroides. |
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| 言語 | en | |||||
| 学位名 | ||||||
| 言語 | ja | |||||
| 学位名 | 博士（医学） | |||||
| 学位授与機関 | ||||||
| 学位授与機関識別子Scheme | kakenhi | |||||
| 学位授与機関識別子 | 16201 | |||||
| 言語 | ja | |||||
| 学位授与機関名 | 香川大学 | |||||
| 言語 | en | |||||
| 学位授与機関名 | Kagawa University | |||||
| 学位授与年月日 | ||||||
| 学位授与年月日 | 2019-07-17 | |||||
| 学位授与番号 | ||||||
| 学位授与番号 | 乙第289号 | |||||
| 権利 | ||||||
| 言語 | en | |||||
| 権利情報 | Copyright © 2016 Elsevier Ltd. All rights reserved. | |||||
| 権利 | ||||||
| 言語 | ja | |||||
| 権利情報 | この博士論文の本文については、次のエルゼビアの著作権ポリシーの規定により公開しています。「博士論文の場合は、エンバーゴ期間に関係なく機関リポジトリに出版社版を公開することができます。」 | |||||
| 権利 | ||||||
| 言語 | en | |||||
| 権利情報 | The text of this doctoral dissertation is published according to the following Elsevier copyright policy. "In the case of doctoral dissertations, publishers can be published in institutional repositories regardless of the Embargo period." | |||||
| 論文ID（NAID） | ||||||
| 識別子タイプ | NAID | |||||
| 関連識別子 | 500001359041 | |||||
| PubMed番号 | ||||||
| 識別子タイプ | PMID | |||||
| 関連識別子 | 27639596 | |||||
| 関連サイト | ||||||
| 関連タイプ | isVersionOf | |||||
| 識別子タイプ | DOI | |||||
| 関連識別子 | https://doi.org/10.1016/j.anaerobe.2016.09.004 | |||||
| 関連サイト | ||||||
| 関連タイプ | isIdenticalTo | |||||
| 識別子タイプ | URI | |||||
| 関連識別子 | https://dl.ndl.go.jp/pid/11344486 | |||||
| 著者版フラグ | ||||||
| 出版タイプ | P | |||||
| 出版タイプResource | http://purl.org/coar/version/c_fa2ee174bc00049f | |||||
| キーワード | ||||||
| 言語 | en | |||||
| 主題Scheme | Other | |||||
| 主題 | Bacteroides | |||||
| キーワード | ||||||
| 言語 | en | |||||
| 主題Scheme | Other | |||||
| 主題 | pheS | |||||
| キーワード | ||||||
| 言語 | en | |||||
| 主題Scheme | Other | |||||
| 主題 | tRNA | |||||
| キーワード | ||||||
| 言語 | en | |||||
| 主題Scheme | Other | |||||
| 主題 | p-chloro-phenylalanine | |||||
| KEID | ||||||
| 値 | 28766 | |||||
