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研究生: 穆可筠
Mu, Ko-Yun
論文名稱: 優化以邏輯閘(AND gate)建構之全細胞生物感測器並檢測苯乙胺
A Whole-Cell Biosensor Based on AND gate system for the Detection of Phenylethylamine
指導教授: 葉怡均
Yeh, Yi-Chun
口試委員: 葉怡均
Yeh, Yi-Chun
蔡伸隆
Tsai, Shen-Long
杜玲嫻
Tu, Ling-Hsien
口試日期: 2023/06/27
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2024
畢業學年度: 112
語文別: 中文
論文頁數: 122
中文關鍵詞: 生物胺全細胞生物感測器苯乙胺
英文關鍵詞: Phenylethylamine, AND gate system, whole-cell biosensor
研究方法: 實驗設計法
DOI URL: http://doi.org/10.6345/NTNU202400576
論文種類: 學術論文
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  • 苯乙胺是一種生物胺,同時也是一種中樞神經系統的興奮物質,通常存在於巧克力和發酵食品中,由於微生物中含有氨基酸脫羧基酶,因此檢測苯乙胺可得知食品的新鮮度。目前,科學家們使用高效液相色譜法、電化學法、螢光材料等儀器對苯乙胺進行檢測,我們實驗室則是使用全細胞生物感測器對苯乙胺進行定量。不過在先前的研究中,苯乙胺感測器並不專一,因此我們對以重組紅螢光蛋白及邏輯閘AND gate系統設計的全細胞生物感測器進行了報導基因、連接子、核醣體結合位點、啟動子的優化。另外,我們也藉由表達氧化酶 (TynA) 和苯乙醛脫氫酶 (FeaB) 蛋白使苯乙胺能夠快速轉化為苯乙醛及苯乙酸,增加檢測環境中苯乙醛和苯乙酸的濃度,使AND gate系統可以更快速的產生重組紅螢光訊號來增加感測器的專一性及優化誘導螢光結果,在研究過程中也找到了最佳的誘導時間和最專一的感測器,並對真實樣品進行初步的檢測,期望開發一個可以實際定量食品中的苯乙胺濃度的全細胞生物感測器。

    Phenylethylamine (PEA) is a biogenic amine and a central nervous system stimulant found in chocolate or fermented foods. Since microorganisms contain amino acid decarboxylase, the freshness of food can be determined by detecting phenethylamine. Currently, scientists use instruments such as HPLC, electrochemical methods, fluorescent materials, etc., to detect phenylethylamine. However, in previous studies from our laboratory, the whole-cell biosensors were not specific to phenethylamine. In this study, we optimized the reporter genes, linkers, ribosome binding sites, and promoters to construct a PEA-specific whole-cell biosensor. We also express the oxidase (TynA) and phenylacetaldehyde dehydrogenase (FeaB) proteins to convert phenylethylamine into phenylacetaldehyde and phenylacetic acid, increase the concentration of phenylacetaldehyde and phenylacetic acid in the environment. After that, AND gate system can generate recombinant red fluorescent proteins more quickly to increase the specificity of the sensor and optimize the induction fold. Furthermore, we determined the best induction time and the most specific sensor, and compared the results with real sample tests. This study has important implications for the convenience of phenethylamine sensing, and the development of this whole-cell biosensor can accurately quantify the concentration of phenylethylamine in food samples.

    誌謝 i 中文摘要 ii ABSTRAT iii 縮寫表 iv 目錄 vi 表目錄 xi 圖目錄 xii Chapter 1 Introduction 1 1-1 生物胺 1 1-1-1 分類及生理現象 1 1-1-2 食品中的生物胺 3 1-1-3 對人體的毒性 4 1-1-4 食品中的生物胺檢測方法 5 1-2 全細胞生物感測器 9 1-2-1 生物感測器 9 1-2-2 大腸桿菌 10 1-2-3 感測機制 11 1-3 苯乙胺 13 1-3-1 代謝途徑 13 1-3-2 檢測方法回顧 14 1-3-3 全細胞生物感測器研究回顧 15 1-4 苯乙胺感測器 21 1-4-1 FeaR/PaaX調控系統 21 1-4-2 邏輯閘 23 1-4-3 重組螢光蛋白與融合標籤 24 1-4-4 感測器設計 25 1-5 研究目的 28 Chapter 2 Materials and Methods 29 2-1 實驗藥品 29 2-2 實驗儀器 33 2-3 基因工程 35 2-3-1 畫盤 (Plate streaking) 35 2-3-2 細菌培養 (Pre-Culture) 36 2-3-3 質體抽取 (Mini-Prep) 37 2-3-4 聚合酶鏈鎖反應 (Polymerase Chain Reaction) 37 2-3-5 膠體電泳 (Gel Electrophoresis) 40 2-3-6 膠體萃取 (Gel Extraction) 42 2-3-7 限制酶切割DNA (restriction Enzyme Digestion) 43 2-3-8 DNA接合反應 (DNA ligation) 44 2-3-9 Golden Gate Assembly 45 2-3-10 KLD 反應 48 2-3-11 轉形作用 (Transformation) 50 2-3-12 Colony PCR check 51 2-3-13 基因定序 52 2-3-14 甘油存菌 (Glycerol stock) 53 2-3-15 建構AND-Gate系統 53 2-4 設計與方法 55 2-4-1 質體設計 55 2-4-2 培養方法 55 2-4-3 誘導 56 2-4-4 螢光分析定量 57 2-4-5 真實樣品前處理 57 2-4-6 真實樣品培養方法 58 2-4-7數據處理 59 Chapter 3 Results and discussions 60 3-1 苯乙胺感測器 60 3-1-1報導基因排列優化 60 3-1-2 Linker長度優化 66 3-1-3 RBS優化 69 3-1-4啟動子優化 70 3-1-5誘導時間優化 72 3-1-6專一性測試 74 3-1-7含tynA及feaB基因之質體建構 77 3-1-8 AND gate系統時間追蹤 81 3-1-9 AND gate系統濃度測試 84 3-1-10 AND gate系統專一性測試 86 3-1-11真實樣品測試 87 Chapter 4 Conclusion 94 Reference 96 附錄 102 附錄1 菌種 102 附錄2 質體 108 附錄3 引子 113 附錄4 質體圖 117

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