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研究生: 陳聖融
SHENG-JUNG CHEN
論文名稱: 利用溶菌型噬菌體改良纖維質體
Improving the Phage-Cellulosome with Lytic Phage
指導教授: 蔡伸隆
SHEN-LONG TSAI
口試委員: 李振綱
Cheng-Kang Lee
葉怡均
Yi-Chun Yeh
學位類別: 碩士
Master
系所名稱: 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年: 2019
畢業學年度: 107
語文別: 中文
論文頁數: 92
中文關鍵詞: 噬菌體纖維質體纖維水解酶釀酒酵母
外文關鍵詞: Phage, Cellulosome, Cellulase, Saccharomyces cerevisiae
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摘要 i Abstract ii 誌謝 iii 總目錄 iv 圖目錄 viii 第一章 緒論 1 1.1研究背景 1 1.2研究動機與目的 1 1.3研究內容 2 第二章 文獻回顧 3 2.1 纖維素 3 2.2 纖維素水解 4 2.3 釀酒酵母 5 2.4 酵母表面展示技術 7 2.5 纖維質體 8 2.6 噬菌體 9 2.7 噬菌體展示技術 10 2.8 λ噬菌體 12 2.9蛋白質交互作用 16 2.10 酵素動力學 18 第三章 材料與實驗步驟 20 3.1 實驗綱要 20 3.2 材料與儀器 21 3.2.1 實驗藥品 21 3.2.2 儀器設備 23 3.2.3 菌種來源 24 3.2.4 引子組 25 3.3 實驗步驟 26 3.3.1 基因轉殖技術 26 3.3.1.1 質體純化法 26 3.3.1.2 聚合酶鏈反應 27 3.3.1.3 瓊脂凝膠電泳與回收 28 3.3.1.4 限制酶酶切作用 30 3.3.1.5 酸接合與化學轉殖法 30 3.3.1.6 電穿孔勝任細胞製備及電穿孔轉殖作用 32 3.3.2 纖維水解酵素之蛋白表達 34 3.3.3 金屬親和層析法 (IMAC) 36 3.3.4 纖維水解酵素活性測定 37 3.3.5 分析方法 38 3.3.5.1 SDS-PAGE凝膠電泳 38 3.3.5.2 DNS還原醣測定法 40 3.3.5.3苯酚-硫酸法 41 3.3.5.4 Bradford蛋白質定量法 42 3.3.6 噬菌體展示技術 44 3.3.6.1噬菌體蛋白表面表達及回收 44 3.3.6.2噬菌體滴度測試 45 3.3.7纖維質體組裝測試 47 3.3.7.1 染色噬菌體組裝於釀酒酵母表面測試 47 3.3.7.2 定量噬菌體於酵母表面的結合數量 48 3.3.7.3 噬菌體纖維質體SH3結合域測試 49 第四章 結果與討論 51 4.1 質體建立 51 4.1.1 噬質體 51 4.1.2 纖維水解酶 60 4.1.2.1 a - 葡聚苷酶 (BGLA) 60 4.1.2.2 內切葡聚醣酶 (celA) 62 4.1.2.3 纖維二醣水解酶 (EC) 63 4.1.3 雙質體之建構 65 4.2 纖維水解酶活性測定 66 4.3 噬菌體殼蛋白之表達 67 4.4 噬菌體纖維質體組裝 68 4.4.1 染色噬菌體組裝於釀酒酵母表面測試 68 4.4.2 噬菌體纖維質體SH3結合域測試 69 4.4.3定量噬菌體於酵母表面的結合數量 71 第五章 結論 72 參考文獻 73

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