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研究生: 陳姵璇
Chen, Pei-Hsuan
論文名稱: 利用微生物 MerR 家族與二元調節系統設計銅離子生物感測器
Construction of MerR-family and two-component system-based whole-cell biosensors for detection of bioavailable copper
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: 2016
畢業學年度: 104
語文別: 中文
論文頁數: 63
中文關鍵詞: 全細胞生物感測器銅離子青枯桿菌耐金屬貪銅菌CueRCopSR紅色螢光蛋白4,5-多巴雙加氧酶甜菜黃色素
英文關鍵詞: whole-cell biosensor, copper, Ralstonia eutropha, Cupriavidus metallidurans, CueR, CopSR, red fluorescence protein, DOPA 4,5-Dioxygenase, betaxanthin
DOI URL: https://doi.org/10.6345/NTNU202204363
論文種類: 學術論文
相關次數: 點閱:198下載:5
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  • 本研究使用微生物抵抗銅離子的調控系統,利用基因重組技術設計量測銅離子的全細胞生物感測器。本研究分別使用青枯桿菌 (Ralstonia eutropha) 中屬於 MerR 家族的 cue 基因調控組,以及耐金屬貪銅菌 (Cupriavidus metallidurans) 中屬於二元調節系統的 cop 基因調控組,以紅色螢光蛋白作為訊號輸出的報導基因來建構質體,設計出不同的銅離子生物感測器。其中,cueR 生物感測器除了能夠量測銅離子之外,在適當的前處理下還能分別量測銀離子與金離子。根據世界衛生組織所公布的飲用水水質準則,飲用水中銅離子的含量不得超過 2 mg/L (31 μM),本研究所設計的 cueR 生物感測器以耐金屬貪銅菌作為質體之宿主時,量測銅離子的最低偵測極限為 25.54 μM,能夠量測飲用水中銅離子是否超標。而在 copSR 生物感測器的實驗中,嘗試使用不同的 cop 啟動子,建構出多種 copSR 生物感測器。也嘗試表現紫茉莉的 4,5-多巴雙加氧酶 (DOPA 4,5-Dioxygenase of Mirabilis jalapa, MjDOD),透過添加 L-多巴 (L-3,4-dihydroxyphenylalanine, L-DOPA) 催化產生出甜菜黃色素 (betaxanthin),以色素的生成作為輸出訊號,進而縮短檢測時間。此外,我們嘗試在不同來源的水樣品中額外添加銅離子進行量測,證明本研究設計的 copSR 生物感測器不會受到水中其他雜質干擾而影響銅離子量測。本研究設計出多種銅離子生物感測器,隨著每種生物感測器偵測範圍不同,可望運用在不同的需求上。

    In this study, we used recombinant DNA technology to develop whole-cell biosensors for the detection of copper ions. The bacterial copper resistance regulons of MerR-family and two-component system (Ralstonia eutropha cue regulon and Cupriavidus merallidurans cop regulon, respectively) were used to regulate the expression of rfp (red fluorescence protein) reporter gene for the construction of whole-cell biosensors. Our results demonstrated that cueR-based biosensor could detect copper, silver, and gold ions under appropriate pretreatments. When using C. metallidurans as a host cell, the limit of detection for copper was 25.54 μM. According to World Health Organization (WHO) guidelines, the recommended value of copper in drinking water is below 2 mg/L (31 μM). The cueR-based copper biosensor designed in this study can successfully detect whether drinking water contains excessive copper ions. In the case of copSR-based biosensors, different copSR-based biosensors were constructed by screening various cop promoters. In order to reduce the detection times, we expressed DOPA 4,5-Dioxygenase of Mirabilis jalapa (MjDOD) which used L-DOPA (L-3,4-dihydroxyphenylalanine) as a substrate to produce yellow fluorescent pigments, betaxanthins. Furthermore, we added known concentrations of copper ions to real water samples, such as tap water and pond water. Our copSR whole-cell biosensor showed no significant sample matrix effect. With various detection ranges and different detection limits, copper biosensors in this study can provide applications with different windows of detection.

    誌謝 i 中文摘要 ii Abstract iii 目錄 iv 圖目錄 vii 表目錄 ix Chapter 1 緒論 1 1.1 第十一族金屬之細胞毒性 1 1.2 大腸桿菌 (E. coli) 對銅離子的排毒機制 2 1.2.1 ComC 蛋白質 2 1.2.2 NADH 脫氫酶-2 (NADH dehydrogenase-2, NDH-2) 3 1.2.3 cue 調控組 (cue regulon) 3 1.2.4 cus 調控組 (cus regulon) 4 1.2.5 pco 調控組 (pco regulon) 5 1.3 全細胞生物感測器 (whole-cell biosensor) 5 1.4 調控蛋白與金屬離子結合機制 6 1.4.1 MerR 家族轉錄調控子 (MerR family transcriptional regulator) 6 1.4.2 二元調節系統 (two component system) 9 1.5 本研究使用之調控蛋白 11 1.5.1 Ralstonia eutropha H16 CueR 11 1.5.2 Cupriavidus metallidurans CH34 CopSR 12 1.6 本研究使用之報導基因 14 1.6.1 紅色螢光蛋白 (red fluorescent protein, rfp) 14 1.6.2 紫茉莉 4,5-多巴雙加氧酶 (DOPA 4,5-Dioxygenase from Mirabilis jalapa, MjDOD) 14 1.7 研究動機與目標 16 Chapter 2 實驗藥品與器材 17 2.1 實驗藥品 17 2.2 實驗儀器 19 Chapter 3 實驗方法 20 3.1 生物感測器質體設計與選殖 (cloning) 步驟 20 3.1.1 cueR 生物感測器之設計 20 3.1.2 copSR 生物感測器之設計 21 3.1.3 分子選殖 (molecular cloning) 24 3.2 實驗步驟 27 3.2.1 cueR 生物感測器之金屬誘導與螢光強度分析 27 3.2.2 copSR 生物感測器之金屬誘導與螢光強度分析 29 Chapter 4 實驗結果與討論 32 4.1 MerR 家族 cueR 生物感測器 32 4.1.1 金屬離子專一性測試 32 4.1.2 劑量反應測試 34 4.1.3 培養基的比較 38 4.1.4 CueR 與CupR 蛋白質的交叉干擾 (crosstalk) 39 4.1.5 銀離子的去除 43 4.1.6 銅離子的去除 44 4.2 二元調節系統 copSR 生物感測器 47 4.2.1 cop 啟動子之銅離子的誘導 47 4.2.2 cop 啟動子之突變 49 4.2.3 銅離子之劑量反應測試 51 4.2.4 金屬離子專一性測試 54 4.2.5 甜菜黃素螢光強度測試 56 4.2.6 真實環境中水樣品之量測 57 Chapter 5 結論 58 參考文獻 60 附錄 A 本研究所使用之菌種、質體與引子序列 A-1 附錄 B 已發表之第十一族元素全細胞生物感測器 B-1

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