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 、CueR 、CopSR 、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:180 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.
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