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研究生： 張謙獻
Zhang, Qian-Xian
論文名稱： 透過基因工程優化全細胞生物感測器：主題一、以CupR蛋白開發的金離子感測器 主題二、以TyrR蛋白開發的酪胺酸感測器
Genetic Engineering to Optimize Whole-cell Biosensor: Part I. CupR-based Sensor for Au(III) Detection. Part II. TyrR-based Sensor for Tyrosine Detection.
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： 2019
畢業學年度： 107
語文別： 中文
論文頁數： 83
中文關鍵詞： 基因工程 、金離子 、耐金屬貪銅菌 、CupR操縱子 、酪胺酸 、TyrR調控組 、全細胞生物感測器
英文關鍵詞： Genetic engineering, Gold ions, Cupriavidus metallidurans, CupR operon, Tyrosine, TyrR regulon, Whole-cell biosensor
DOI URL： http://doi.org/10.6345/NTNU201900430
論文種類： 學術論文
相關次數： 點閱：134  下載：0 
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    • 全細胞生物感測器在感測小分子化合物時具有極大的優勢，能夠使用簡單且低成本的裝置達成原本需要利用貴重儀器才能達到的靈敏度，並且有專一強、對培養環境需求低等優點。在本研究中主要使用基因工程優化全細胞生物感測器，使其能夠分別對金離子及酪胺酸進行感測。在金離子感測器的方面，我們運用Cupriavidus metallidurans中的CupR蛋白作為調節機制，使用專一性蛋白結合序列對報導的螢光基因進行表現。藉由運用同屬MerR家族但不同蛋白的專一性蛋白結合序列，探討其在細胞內與蛋白結合能力。對於優化 TyrR 作為調控蛋白的酪胺酸全細胞生物感測器，使用了細菌代謝芳香族胺基酸的重要基因組TyrR 調控組作為調控機制，利用了兩個分別響應培養物中不同酪胺酸濃度的啟動子，控制紅色及綠色螢光信號的輸出。最後開發了一種無須額外添加其餘試劑的量測平台，讓使用者只需要加入樣品，就可藉由量測數據得知樣品中酪胺酸濃度。

    The whole-cell biosensor has great advantages in sensing small molecule compounds. It can achieve sensitivity through a simple setup and low-cost device instead of using expensive instruments. Furthermore, it has the advantages of great specificity and low demand for the cultivated environment. In this research, genetic engineering was used to optimize the whole-cell biosensor to enable sensing of gold ions and tyrosine, respectively. For gold ion sensors, we used the CupR protein, which was adopted from Cupriavidus metallidurans, as our regulatory protein mechanism to express the fluorescent proteins. To investigate the binding ability between the protein binding sequences and CupR in the cells, we changed the CupR binding sequences of the promoters. For the development of tyrosine whole-cell biosensor, we used TyrR as a regulatory protein. TyrR regulator plays an important role in bacteria for the metabolization of aromatic amino acids. Two tyrosine-responsive promoters are used to differentially control the production of red and green fluorescent proteins signals in response to tyrosine levels in culture. To make the device more user-friendly, we developed a reagentless platform in which biocompatible agarose is used as an entrapment agent with cell sensors and growth media mounted within the gel matrix.

    致謝 I 中文摘要 II Abstract III 目錄 IV 圖目錄 VII 表目錄 X 第一章 緒論 1 1 基因工程 (Genetic engineering) 1 2 全細胞生物感測器 (Whole-cell biosensor) 2 2-1 大腸桿菌 (Escherichia coli) 3 2-2 耐金屬貪銅菌 (Cupriavidus metallidurans) 4 2-3 MerR家族轉錄調控子 (MerR family transcriptional regulator) 4 2-4 TyrR調控組 (TyrR regulon) 9 2-5 核糖體結合位點 (Ribosome-binding site, RBS) 13 3 第十一族金屬 14 4 酪胺酸 (Tyrosine, Tyr) 15 5 報導基因 (reporting gene) 17 5-1 紅色螢光蛋白 (Red Fluorescent Protein, RFP) 18 5-2 綠色螢光蛋白 (Green Fluorescent Protein, GFP) 18 6 研究動機與目標 19 6-1 金離子生物感測器 19 6-2 酪胺酸生物感測器 21 第二章 實驗藥品與器材 23 1 實驗藥品 23 2 實驗儀器 25 第三章 實驗方法 26 1 分子選殖 (Molecular cloning) 26 1-1 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 27 1-2 引子黏合 (Annealing) 28 1-3 載體 (Vector) 29 1-4 限制內切酶裁切 (Restriction enzyme digestion) 29 1-5 接合作用 (Ligation) 30 1-6 轉形作用 (Transformation) 31 1-7 定序 (sequencing) 31 1-8 菌種儲存 (glycerol store) 32 1-9 接合作用 (conjugation) 32 2 金離子生物感測器之設計 33 2-1 質體設計 (Plasmid design) 33 2-2 啟動子設計 (Promoter design) 35 3 酪胺酸生物感測器之設計 37 4 實驗步驟 38 4-1 金離子感測器 38 4-2 酪胺酸感測器 40 4-3 螢光強度與微生物OD600生長分析 44 4-4 數據處理及偵測極限計算 45 第四章 實驗結果與討論 46 1 金離子感測器 46 1-1 反應性測試 46 1-2 MerR家族蛋白結合位置測試 47 1-3 RNA聚合酶作用片段對結合能力的影響 48 1-4 專一性結合位點突變比較 49 1-5 分屬不同MerR家族蛋白專一性結合位置能力測試 50 1-6 比較作為基礎啟動子的PcupC與PcupA 52 1-7 以雙質體系統提升CupR濃度對螢光的影響 54 2 酪胺酸感測器 55 2-1 反應性測試 55 2-2 專一性測試 56 2-3 核醣體結合位比較 57 2-4 轉譯TyrR之啟動子能力測試 59 2-5 時間追蹤 60 2-6 更換核醣體結合位測試 61 2-7 稀釋條件測試 62 2-8 模擬真實樣品環境測試 64 2-9 便攜裝置應用測試 65 第五章 結論 67 附錄 70 1 引子 70 2 質體 73 3 菌種 78 參考文獻 80

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