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研究生: 徐儷文
Hsu, Li-Wen
論文名稱: 使用全細胞生物感測器在微流道設備中同時測定L-苯丙胺酸、苯乙胺和苯乙酸
Simultaneous Determination of L-phenylalanine, Phenylethylamine, and Phenylacetic Acid Using Whole-cell Biosensors within a Microchannel Device
指導教授: 葉怡均
Yeh, Yi-Chun
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2020
畢業學年度: 108
語文別: 中文
論文頁數: 83
中文關鍵詞: 苯丙酮尿症全細胞生物感測器微流道設備苯丙胺酸苯乙胺苯乙酸
英文關鍵詞: phenylketonuria (PKU), whole-cell biosensor, microchannel devices, phenylalanine (Phe), phenylethylamine (PEA), phenylacetic acid (PA)
DOI URL: http://doi.org/10.6345/NTNU202000699
論文種類: 學術論文
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  • 誌謝 i 中文摘要 ii ABSTRACT iii 縮寫表 iv 目錄 v 表目錄 ix 圖目錄 x Chapter 1 緒論 (Introduction) 1 1-1 苯丙酮尿症 (Phenylketonuria, PKU) 1 1-1-1 人體中芳香族胺基酸代謝 1 1-1-2 苯丙酮尿症的治療與管理 3 1-2 全細胞生物感測器 (Whole cell-based biosensor) 4 1-2-1 大腸桿菌 (Escherichia coli) 5 1-2-2 芳香族化合物代謝與調控 6 1-2-2-1 苯乙胺代謝調控組-mao operon 7 1-2-2-2 苯乙酸代謝調控組-paa operon 8 1-2-3 芳香族胺基酸調控組 10 1-2-3-1 TyrR regulon 10 1-2-3-2 啟動子的活化 12 1-2-3-3 啟動子的抑制 13 1-2-4 核醣體結合位點 (Ribosome binding site, RBS) 15 1-3 碳源代謝抑制效應 (Carbon catabolite repression, CCR) 17 1-3-1 磷酸根轉移系統 17 1-3-2 葡萄糖分解代謝抑制系統 18 1-4 報導基因 (Reporter gene) 20 1-4-1 紅色螢光蛋白 (Red Fluorescent Portein, RFP) 21 1-4-2 黃色螢光蛋白 (Yellow Fluorescent Portein, YFP) 21 1-4-3 綠色螢光蛋白 (Green Fluorescent Portein, GFP) 22 1-4-4 青色螢光蛋白 (Cyan Fluorescent Portein, CFP) 22 1-4-5 螢光蛋白的多重標記 (Multiple labeling) 23 1-5 其他檢測 PKU 的技術 24 1-5-1 PKU患者檢測方法 24 1-5-2 基於全細胞感測之檢測法 27 1-6 實驗動機與目的 (Motivation and procedure) 29 Chapter 2 實驗方法與設計 (Experimental design) 30 2-1 實驗儀器 30 2-2 實驗藥品 31 2-3 基因克隆 (Gene Cloning) 33 2-3-1 質體抽取 (plasmid extraction) 33 2-3-2 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 33 2-3-3 限制酶切割 (restriction enzyme digestion) 35 2-3-3 接合 (ligation) 35 2-3-4 轉化 (transformation) 36 2-3-5 質體檢查 (PCR check) 36 2-3-7 膠體萃取/聚合酶連鎖反應清理 (gel extraction/PCR clean up) 37 2-3-8 存菌 (bacteria storage) 37 2-3-9 勝任細胞製作 (competent cell) 38 2-4 實驗方法 39 2-4-1 質體設計 39 2-4-2 培養條件 40 2-4-3 誘導試劑 (inducer) 41 2-4-5 螢光分析定量 41 2-4-6 螢光顯微鏡 42 2-4-7 混合菌液 42 2-4-8 真實樣品 43 2-4-9 數據處理 44 2-4-10 微流道之操作方法 44 Chapter 3 實驗結果與討論 45 3-1 啟動子轉錄條件優化 45 3-1-1 IPTG 濃度優化 46 3-1-2 誘導時間優化 48 3-2 RBS 選擇 49 3-3 螢光蛋白挑選 51 3-4 劑量反應及偵測極限 53 3-4-1 Phe 感測器 (YCY_1065) 54 3-4-2 PEA 感測器 (YCY_1062) 54 3-4-3 PA 感測器 (YCY_1051) 55 3-5 混合菌液 56 3-5-1 M9 medium 56 3-5-2 微流道應用 57 3-5-3 真實樣品測試 58 3-6 雙信號輸出之全細胞生物感測器 (YCY_1070) 59 3-6-1 Phe 及 PA 之劑量反應 60 3-6-1 干擾性測試 61 3-6-2 真實樣品測試 62 3-6-3 葡萄糖影響 63 3-6-4 YCY_1070 質體設計改良 64 3-6-4-1 tyrP 啟動子的突變 64 3-6-4-2 三信號輸出之全細胞生物感測器 (Tyr+Phe+PA) 66 Chapter 4 結論 70 References 71 附錄 75 附錄1 菌種 75 附錄2 質體 79 附錄3 引子 83

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