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

Abstract

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.