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研究生: 趙貽
Yi Chao
論文名稱: 噬菌體輔助自主連續定向演化之系統建立
A system for Phage Assisted Autonomous Continuous Directed Evolution
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: 99
中文關鍵詞: 定向演化胞內定向演化M13噬菌體濃度梯度盤
外文關鍵詞: Directed evolution, In vivo directed evolution, M13 phage, Concentration gradient plate
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摘要 iii Abstract iv 致謝 v 總目錄 vi 表目錄 ix 圖目錄 x 縮寫表 xii 第一章 緒論 1 1.1 研究背景 1 1.2 研究動機與目的 2 1.3 研究內容 3 第二章 文獻回顧 5 2.1 定向演化 5 2.2 噬菌體輔助連續演化 7 2.3 細胞溝通 8 2.4 噬菌體輔助技術 10 2.4.1 M13噬菌體簡介 11 2.4.2 M13噬菌體之gIII殼蛋白 12 2.5 附屬質體 13 2.5.1 細菌趨向性蛋白:CheZ 13 2.5.2 綠色螢光蛋白GFP 15 2.6 胞內誘變質體 16 2.6.1 降低校對機制:dnaQ962蛋白 17 2.6.2 破壞錯位修復:dam酶、SeqA結合域 18 2.6.3 減少鹼基修復:cda1蛋白、ugi蛋白 19 2.6.4 提高胞內致突變物質:emrR轉錄調控子 19 2.7 奈米螢光素酶(Nano-Luciferase) 20 第三章 實驗材料與方法 21 3.1 菌種與質體 21 表3.1 菌種與質體的來源或基因體 21 3.2 實驗藥品 21 表3.2 藥品與材料 21 3.3 實驗器材 24 表3.3 實驗儀器 24 3.4 實驗流程 25 3.5 質體DNA純化法(Mini-prep) 26 3.6 聚合酶連鎖反應(Polymerase Chain Reaction, PCR) 27 表3.4 選擇噬質體引子序列 28 表3.5 大腸桿菌引子序列 28 表3.6 附屬質體引子序列 29 表3.7 報告質體引子序列 30 表3.8 聚合酶連鎖反應溶液 30 表3.9 聚合酶連鎖反應實驗條件 31 3.7 DNA瓊脂凝膠電泳(DNA Agarose gel electrophoresis) 31 3.8 DNA瓊脂凝膠回收(DNA recovery) 32 3.9 酶切(Digestion) 33 表3.10 酶切法反應溶液 33 3.10 核酸接和作用(DNA ligation) 33 表3.11 核酸接合反應溶液 34 3.11 勝任細胞(Competent cell)製備 34 3.12 轉殖作用(Transform) 35 3.13 細胞接合作用(Conjugation) 36 3.14 電穿孔勝任細胞製備及轉殖作用 37 3.15 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(SDS-PAGE) 38 3.16 西方墨點分析法 40 3.17 噬菌體組裝及回收 41 3.18 噬菌體滴度測試 43 3.19 濃度差半固態盤之製備及演化 44 3.20 濃度差半固態盤之回收 45 3.21 奈米熒光素酶試驗(Nano-Luciferase Assay) 46 3.22 點突變重疊延伸聚合酶連鎖反應 47 表3.12 點突變重疊延伸引子序列 48 第四章 結果與討論 50 4.1選擇噬菌體(Selection Phage, SP)之建構 50 4.2附屬質體(Accessory Plasmid, AP)之建構 52 4.2.1 附屬質體AP-pET(xf1)-pUCori-CheZ-gIII-GFP 52 4.2.2 附屬質體AP-pET(xf1)-pUCori-CheZ-GFP-gIII 54 4.2.3 附屬質體Ap-pET(xf1)-pUCori-pT7/T3-CheZ-gIII-GFP和Ap-pET(xf1)-pUCori-pT7T3-CheZ-GFP-gIII之建構 56 4.2.4 附屬質體Ap-pET(xf1)- pUCori-pT3-CheZ-gIII–GFP、Ap-pET(xf1)-pUCori-pT3- CheZ-GFP-gIII之建構 57 4.3報告質體(Report Plasmid, RP)之建構 58 4.3.1報告質體RP-pT7-Nluc-ptac和RP-pT3-Nluc-ptac之建構 59 4.3.2報告質體RP-pT7-Nluc-ptac-T7RNAP Library和RP-pT3-Nluc-ptac-T7RNAP Library之建構 61 4.4正控制報告質體RP-T7RNAP E222K/N748D建構 63 表4.1 變異種T7RNAP E222K/N748D胺基酸序列 63 4.5宿主細胞的接合作用 66 4.6重組噬菌體的滴度和感染分析 67 4.7本系統於SDS-PAGE和西方墨點法之分析 69 4.8半固態盤 71 4.9基因庫序列分析 74 表4.2 第一次演化定序結果 75 表4.3 第二次演化定序結果 76 4.10正控制組定序及奈米螢光素酶試驗分析 77 表4.4 正控制變異種定序結果 78 4.11基因庫奈米螢光素酶試驗 78 第五章 結論 81 第六章 參考文獻 82

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