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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 徐丞志 | zh_TW |
| dc.contributor.advisor | Cheng-Chih Hsu | en |
| dc.contributor.author | 林泓君 | zh_TW |
| dc.contributor.author | Hung-Chun Lin | en |
| dc.date.accessioned | 2024-09-25T16:34:55Z | - |
| dc.date.available | 2024-09-26 | - |
| dc.date.copyright | 2024-09-25 | - |
| dc.date.issued | 2024 | - |
| dc.date.submitted | 2024-09-02 | - |
| dc.identifier.citation | 1. Li, D.; Wang, P.; Wang, P.; Hu, X.; Chen, F., The gut microbiota: A treasure for human health. Biotechnol. Adv. 2016, 34 (7), 1210-1224.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/96006 | - |
| dc.description.abstract | 腸道微生物群中的雙歧桿菌屬(Bifidobacterium)被視為益生菌，與人類健康息息相關，不僅能夠改善腸道健康，還能增強免疫力。為了深入探討雙歧桿菌是如何影響人類宿主、釐清是否有特定分子在此扮演重要角色，針對雙歧桿菌特定基因和代謝路徑的調控至關重要。然而，雙歧桿菌內限制修飾(RM)系統會將外來未甲基化的DNA切除，造成轉形(transformation)效率大幅降低，導致難以在雙歧桿菌屬中開發基因工程技術。本篇論文中，我們成功在雙歧桿菌屬中建立了一系列CRISPR-Cas9基因鹼基編輯器(cytosine base editor systems, cBEST)，藉由精準調控鹼基編輯器和sgRNA的表現量來編輯雙歧桿菌屬中特定的基因。為了解決轉形效率差的問題，我們在龍根菌(Bfidobacterium longum NCIMB 8809)以及青春雙歧桿菌(Bifidobacterium adolescentis DSM 20083)中利用cBEST技術消除限制酶或甲基化特定DNA序列來躲避限制修飾系統，我們成功將轉型效率提高至三十萬倍，而基因編輯成功率則提高至100%。接著，為了驗證cBEST方法能精準調控特定的代謝路徑，我們透過分析不同變異株的甲硫胺酸(methionine)循環路徑，了解DNA甲基化與甲硫腺苷(5’-methylthioadenosine)調控的關係。我們也成功在不同雙歧桿菌屬中編輯膽鹽水解酶(bsh)，並透過LC-MS/MS定量分析，證明我們能精準調控雙歧桿菌屬的膽酸代謝路徑。整體而言，我們成功利用CRISPR-Cas9基因編輯技術在雙歧桿菌屬中精準調控代謝途徑，並提供策略去躲避限制修飾系統，我們相信此方法必能成為研究腸道微生物強而有力的工具。 | zh_TW |
| dc.description.abstract | Intestinal microbiota members of the Bifidobacterium genus are increasingly recognized as probiotic potential and therapeutics applications to improve human health. However, the paucity of genetic tools and the prevalence of restriction modification (RM) systems limit the genetic manipulation in Bifidobacterium. In this study, we established a series of CRISPR/Cas9 cytosine base editor systems (cBESTs) with fine tuning base editor and sgRNA expression that are portable across multiple Bifidobacteria. We showed that bypassing RM systems by either eliminating restriction endonucleases or matching DNA methylation patterns in B. longum NCIMB 8809 and B. adolescentis DSM 20083 significantly improved both transformation (up to 300,000-fold) and editing efficiencies (up to 100%). Furthermore, we successfully manipulated methionine cycle pathway to elucidate the correlation between DNA methylation and 5’-methylthioadenosine regulation through MetK-deficient and RM-disrupted strains. Finally, we achieved the genetic knockout of the conserved bile salt hydrolase (bsh) gene in B. longum NCIMB 8809, B. longum DSM 20219 and B. infantis DSM 20088, effectively deactivating bile acids deconjugation. In summary, the ability to efficiently engineer Bifidobacterium genomes will definitely open new avenues for research and applications towards improving human health. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2024-09-25T16:34:55Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2024-09-25T16:34:55Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員會審定書 i
誌謝 ii 摘要 iv Abstract v 圖次 ix 表次 xii Chapter 1. Introduction 1 1-1. Bifidobacterium 1 1-2. CRISPR-Based Genome Editing Methods for Bacteria 2 1-3. Restriction Modification Systems 4 1-4. Research Objectives 6 Chapter 2. Materials and Methods 7 2-1. Chemicals and Reagents 7 2-2. Bacterial Strains and Culturing 7 2-3. Protospacer Design for Gene Knockout 7 2-4. Electroporation of Bifidobacterium Strains 8 2-5. Determination of Transformation Efficiencies 10 2-6. Validation of Genome Editing and Determination of Genome Editing Efficiencies as well as Gene Editing Windows 10 2-7. Plasmid Curing from Bifidobacterium Transformants 11 2-8. mCherry Reporter Assay for Promoter Screening and Characterization 11 2-9. Bacterial Plate Cultures for Monitoring Methionine Cycle Pathway 12 2-10. Metabolomics Using LC-QE Analysis for Bacterial Plate Cultures 13 2-11. Targeted Methionine Derived Metabolites Using LC-QqQ Analysis 14 2-12. Bacterial Cultures and Quantification Methods for Bile Acids Production 17 2-13. Statistical Analysis 20 Chapter 3. Results 21 3-1. Promoter Characterization in Bifidobacteria 21 3-2. Cytosine Base Editor Systems (cBESTs) in B. longum NCIMB 8809 24 3-3. Enhanced Transformation and Gene Editing Efficiencies in RM-Disrupted Strains Derived from B. longum NCIMB 8809 and B. adolescentis DSM 20088 28 3-4. Monitoring Methionine Cycle Pathway in RM-Disrupted Strains 36 3-5. Unique RM Systems across Bifidobacterium Strains 41 3-6. Highly Efficient and Streamlined Genome Editing in RM-Disrupted Strains 43 3-7. Identical cBEST Constructs for Metabolic Regulations in Bifidobacterium spp. 48 Chapter 4. Discussion & Conclusion 52 Chapter 5. References 56 Chapter 6. Supporting Information 63 | - |
| dc.language.iso | en | - |
| dc.title | 針對雙歧桿菌屬開發基因編輯技術以調控代謝途徑：破壞限制修飾系統促進基因編輯效率 | zh_TW |
| dc.title | Development of CRISPR-Cas9 Base Editing Tools for Metabolically Engineerable Bifidobacteria: Disruption of Restriction-Modification Systems Facilitates Gene Editing Efficiency | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-1 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 朱忠瀚;林建達;張明姿 | zh_TW |
| dc.contributor.oralexamcommittee | Chung-Han Chu;Jian-Da Lin;Mingzi M. Zhang | en |
| dc.subject.keyword | 基因編輯,CRISPR-Cas9,雙歧桿菌,限制修飾系統,DNA甲基化,甲硫腺苷,膽鹽水解酶, | zh_TW |
| dc.subject.keyword | Genome editing,CRISPR-Cas9,Bifidobacterium,Restriction-modification systems,DNA methylation,5’-methylthioadenosine,Bile salt hydrolase, | en |
| dc.relation.page | 87 | - |
| dc.identifier.doi | 10.6342/NTU202404344 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2024-09-04 | - |
| dc.contributor.author-college | 理學院 | - |
| dc.contributor.author-dept | 化學系 | - |
| dc.date.embargo-lift | 2025-09-01 | - |
| 顯示於系所單位： | 化學系 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-113-1.pdf | 11.78 MB | Adobe PDF | 檢視/開啟 |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。