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研究生: 劉建平
Liu, Jian-Pin
論文名稱: 優化全細胞生物感測器對苯丙胺酸與食品檢測之應用
Genetic Engineering to Optimize Whole-cell Biosensor Toward Phenylalanine for Food Determination applications
指導教授: 葉怡均
Yeh, Yi-Chun
口試委員: 杜玲嫻
Tu, Ling-Hsien
蔡伸隆
Tsai, Shen-Long
葉怡均
Yeh, Yi-Chun
口試日期: 2022/07/05
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2022
畢業學年度: 110
語文別: 中文
論文頁數: 121
中文關鍵詞: 苯丙酮尿症全細胞生物感測器苯丙胺酸食品檢測
英文關鍵詞: phenylketonuria (PKU), whole-cell biosensor, phenylalanine (Phe), food analysis, soft drinks
DOI URL: http://doi.org/10.6345/NTNU202200974
論文種類: 學術論文
相關次數: 點閱:101下載:0
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  • 中文摘要 i ABSTRACT ii 縮寫表 iii 目錄 v 圖目錄 ix 表目錄 xiii Chapter 1緒論 1 1-1全細胞生物感測器 (Whole-cell biosensor) 1 1-1-1生物感測器 1 1-1-2 大腸桿菌 2 1-1-3 熱誘導 4 1-2 芳香族胺基酸調控 5 1-2-1 芳香族胺基酸在大腸桿菌體內合成路徑 5 1-2-2 TyrR regulon 7 1-2-3 Activation 10 1-2-4 Repression 11 1-3 苯丙酮尿症 (Phenylketonuria) 13 1-3-1 胺基酸代謝疾病 13 1-3-2 苯丙酮尿症 13 1-3-3 治療方法 15 1-3-4 檢測方法回顧 17 1-4 苯乙胺代謝與其衍伸藥物檢測 21 1-4-1 苯乙胺代謝途徑 21 1-4-2 苯乙胺衍伸藥物-安非他命 22 1-4-3 檢測方法 25 1-5 實驗動機與目的 30 Chapter 2 實驗方法與設計 32 2-1 實驗儀器 32 2-2 實驗藥品 34 2-3 基因工程 38 2-3-1 畫盤 (Plate streaking) 38 2-3-2 抽取質體 (Plasmid extraction) 39 2-3-3 聚合酶鏈鎖反應 (Polymerase Chain Reaction, PCR) 39 2-3-4 膠體電泳 (Gel electrophoresis)與膠體萃取 (Gel extraction) 41 2-3-5 限制酶切割 (restriction enzyme digestion) 42 2-3-6 接合 (Ligation) 43 2-3-7 Golden Gate Assembly 44 2-3-8 突變 (Mutagenesis) 46 2-3-9 轉形作用 (transformation) 47 2-3-10 質體檢查 (PCR check) 48 2-3-11 定序 (Sequencing) 49 2-3-12 存菌 (Glycerol stock) 49 2-3-13 雙質體系統建構 49 2-4 苯丙酮尿症生物感測器設計與實驗方法 50 2-4-1 啟動子設計 50 2-4-2 培養過程 51 2-4-3 誘導試劑 51 2-4-4 螢光分析定量 52 2-4-5 真實樣品前處理 52 2-4-6 真實樣品培養條件 54 2-4-7 數據處理 55 2-5 苯乙胺衍生藥物感測器開發 55 2-5-1 質體設計 55 2-5-2 突變條件 55 2-5-3 培養條件 57 Chapter 3 實驗結果與討論 58 3-1 苯丙胺酸感測器 58 3-1-1 PtyrP啟動子突變 58 3-1-2 TyrR蛋白優化 60 3-1-3 PRPL 啟動子系統 63 3-1-5 IPTG誘導濃度優化 65 3-1-6雙訊號系統優化 66 3-1-7 mTCG質體優化 69 3-1-8 雙質體系統設計與比較 70 3-1-9 誘導時間優化 72 3-1-10 雙質體系統與單質體系統的比較 75 3-1-11 真實樣品測試 79 3-2 苯乙胺感測器 83 3-3 苯乙胺衍伸物感測器開發 85 3-3-1 培養條件優化與感測器選擇 85 3-3-2 buffer termination 建構 87 3-3-3 抗抗生素質體建構 89 Chapter 4 結論 92 附錄 94 附錄1 菌種 94 附錄2 質體 102 附錄3 引子 112 Reference 115

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