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研究生: 黃宜蓁
Yi-Chen Huang
論文名稱: 趨磁細菌基因轉殖技術開發及降解有機磷農藥之應用
Development of Magnetotactic Bacteria Gene Transfer Technology and Application in Degrading Organophosphorus Pesticides
指導教授: 蔡伸隆
Shen-Long Tsai
口試委員: 李振綱
Cheng-Kang Lee
王勝仕
Sheng-Shih Wang
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: 2021
畢業學年度: 109
語文別: 中文
論文頁數: 103
中文關鍵詞: 趨磁細菌
外文關鍵詞: Magnetotactic Bacteria
相關次數: 點閱:133下載:0
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  • 目錄 碩士學位論文指導教授推薦書 I 碩士學位考試委員審定書 II 摘要 III Abstract IV 致謝 V 目錄 VI 圖目錄 IX 表目錄 XI 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 4 2.1 趨磁細菌 Magnetotactic bacteria 4 2.1.1 趨磁細菌之介紹 4 2.1.2 磁小體生成機制 6 2.1.3 磁小體表達之應用 8 2.1.4 趨磁細菌表達之載體 9 2.2 固定化酶技術 10 2.3 有機磷水解酶 OPH 11 2.4 綠螢光蛋白 GFP 12 第三章 實驗材料與方法 13 3.1 菌種與質體 13 3.2 實驗藥品 14 3.3 實驗器材 18 3.4 質體建構 19 3.4.1 菌種培養 19 3.4.2 質體純化法 22 3.4.3 聚合酶連鎖反應 26 3.4.4 瓊脂凝膠電泳 32 3.4.5 基因序列回收 33 3.4.6 限制酶酶切反應 34 3.4.7 核酸接合作用 35 3.4.8 大腸桿菌勝任細胞製備 36 3.4.9 大腸桿菌之轉型作用 38 3.4.10 大腸桿菌電穿孔勝任細胞製備 39 3.4.11 大腸桿菌之電穿孔轉型作用 41 3.4.12 菌種保存 42 3.5 菌種間的接合作用 43 3.6 有機磷水解酶活性測試 45 3.7 綠螢光蛋白螢光強度測試 46 3.8 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 SDS-PAGE 47 3.9 顯微鏡觀測 50 第四章 結果與討論 51 4.1 趨磁細菌 Magnetospirillum gryphiswaldense,MSR-1 51 4.2 組成型融合蛋白表達 53 4.2.1 錨蛋白MamC之擴增 53 4.2.2 pET-MamC-OPH質體建構 54 4.3 趨磁細菌之基因轉殖技術開發 56 4.3.1 pBAM1-MamC-OPH質體建構 58 4.3.2 pBAM1-PmamDC50-OPH質體建構 60 4.3.3 pBAM1-PmamDC50-GFP質體建構 63 4.3.4 pBAM1-PmamDC316-OPH質體建構 65 4.3.5 pBAM1-PmamDC316-GFP質體建構 68 4.3.6 pBAM1-Pmsp3-OPH質體建構 70 4.3.7 pBAM1-Pmsp3-GFP質體建構 74 4.4 趨磁細菌之接合作用 76 4.5 蛋白質分析 SDS-PAGE 77 4.6 有機磷水解酶蛋白活性測試 79 4.6.1 繪製對硝基苯酚之檢量線 79 4.6.2 OPH活性測試 80 4.7 綠螢光蛋白螢光強度測試 81 第五章 總結 82 第六章 參考文獻 83 第七章 附錄 86   圖目錄 圖1.1 實驗流程圖 3 圖2.1 趨磁細菌內磁小體之透射電子顯微鏡圖 5 圖2.2 磁小體生物合成機制 7 圖2.3 優化載體與染色體整合示意圖 10 圖4.1 MSR-1生長曲線 51 圖4.2 MSR-1於不同氣體條件下之固態培養 52 圖4.3 MSR-1顯微觀測圖 52 圖4.4 組成型融合蛋白表達序列 53 圖4.5 pET-MamC-OPH質體圖 54 圖4.6 pET-MamC-OPH之Colony PCR結果 55 圖4.7 pET-CI-OPH與pET-MamC-OPH酶切確認圖 55 圖4.8 pir基因之PCR結果 57 圖4.9 pBAM1-MamC-OPH質體圖 58 圖4.10 pBAM1-MamC-OPH之Colony PCR結果 59 圖4.11 pBAM1-MamC-OPH之酶切確認圖 59 圖4.12 pBAM1-PmamDC50-OPH質體圖 61 圖4.13 pBAM1-PmamDC50-OPH之Colony PCR結果 62 圖4.14 pBAM1-PmamDC50-OPH之酶切確認圖 62 圖4.15 pBAM1-PmamDC50-GFP質體圖 63 圖4.16 pBAM1-PmamDC50-GFP之Colony PCR結果 64 圖4.17 pBAM1-PmamDC50-GFP之酶切確認圖 64 圖4.18 pBAM1-PmamDC316-OPH之質體圖 66 圖4.19 pBAM1-PmamDC316-OPH之Colony PCR結果 67 圖4.20 pBAM1-PmamDC316-OPH之酶切確認圖 67 圖4.21 pBAM1-PmamDC316-GFP之質體圖 68 圖4.22 pBAM1-PmamDC316-GFP之Colony PCR結果 69 圖4.23 pBAM1-PmamDC316-GFP之酶切確認圖 69 圖4.24 pUC18- Pmsp3-MamC之質體圖 70 圖4.25 pUC18- Pmsp3-MamC之Colony PCR結果 71 圖4.26 pUC18- Pmsp3-MamC之酶切確認圖 71 圖4.27 pBAM1-Pmsp3-OPH之質體圖 72 圖4.28 pBAM1-Pmsp3-OPH之Colony PCR結果 73 圖4.29 pBAM1-Pmsp3-OPH之酶切確認圖 73 圖4.30 pBAM1-Pmsp3-OPH之質體圖 74 圖4.31 pBAM1-Pmsp3-OPH之Colony PCR結果 75 圖4.32 pBAM1-Pmsp3-OPH之酶切確認圖 75 圖4.33 SM10與MSR-1之生長曲線 76 圖4.34 OPH蛋白表達分析圖 77 圖4.35 GFP蛋白表達分析圖 78 圖4.36 有機磷水解酶之降解機制 79 圖4.37 對硝基苯酚(p-nitrophenol)之檢量線 79 圖4.38 有機磷水解酶活性測試結果 80 圖4.39 不同GFP融合蛋白之單位螢光強度 81   表目錄 表3.1 本實驗菌種介紹 13 表3.2 本實驗質體介紹 13 表3.3 藥品清單 14 表3.4 設備清單 18 表3.5 FSM培養液 20 表3.6 ACA培養盤 21 表3.7 PCR反應溶液 27 表3.8 PCR反應參數設定 27 表3.9 本研究設計之引子 28 表3.10 酶切反應溶液 34 表3.11 核酸接合反應溶液 35 表3.12 Inoue Transformation Buffer溶液 36 表3.13 OPH反應溶液 45 表3.14 SDS-PAGE膠體溶液 48 表3.15 10x SDS running buffer溶液 49 表3.16 5x Sample buffer溶液 49 表7.1 磁小體相關蛋白列表 86

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