以邏輯閘建構全細胞生物感測器檢測銅離子
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2023
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全細胞生物感測器是利用細菌作為感測器主體，透過基因工程技術，藉由賦予它不同調控基因組，使之具有檢測特定待測物的能力，其具有操作方便、價格低廉，對環境污染低等優點，使全細胞生物感測器越來越蓬勃發展。本論文是利用耐金屬貪銅菌 (Cupriavidus metallidurans, C. metallidurans) 作為宿主細菌，並且融入邏輯閘中的AND gate概念設計質體，希望利用兩個啟動子PCopA (Cu2+、Zn2+、Cd2+) 與PCopZ (Cu2+、Au3+、Ag+) 之間的交集，提高對銅離子的專一性，並且使用了Spy Tag/Catcher黏合標籤，提高重組sfCherry3C(1-10)、sfCherry3C(11)的效率以及重組T7 RNA聚合酶 (拆成C-T7和N-T7兩片段) 並作為訊號放大器，提高檢測銅離子的表現。在以上兩個系統，皆已成功建構出對銅離子專一的菌株，在兩系統中: sfCherry3C (2.5-250 μM) 、T7 RNAP (0.1-5 μM) 都有良好的線性，以及低偵測極限，但目前對於背景值以及檢測倍率的方面還需做進一步的優化。結論來說，我成功了開發了一個對銅離子專一的全細胞生物感測器，雖然目前的檢測表現還有進步空間，但可以利用此兩系統作為基礎，優化並發展出更完善的檢測器。
Whole-cell biosensors (WBCs) utilize bacteria as hosts for detection. In order to detect environmental pollutants such as metal ions, I utilized genetic engineering techniques to introduce genes into bacteria to achieve our goal. WBCs are known for their low cost, simplicity, and environmentally friendly, which contribute to their rapid development. In my research, I employed C. metallidurans as the host organism and integrated the concept of an AND gate into plasmid design. I chose two promoters, PCopA (Cu2+, Zn2+, Cd2+) and PCopZ (Cu2+, Au3+, Ag+), to enhance the specificity of Cu2+ detection. Additionally, I introduced the recombinant tag Spy Tag/Catcher to improve the efficiency of complementation sfCherry3C(1-10), sfCherry3C(11), and fusion T7 RNA polymerase (C-T7/N-T7), serving as a signal amplifier to enhance Cu2+ detection performance. I successfully constructed Cu2+-specific strains in both of systems. The sfCherry3C system and T7 RNAP system exhibited good linear ranges of 2.5-250 μM and 0.1-5 μM, respectively. Despite their low limit of detection. Unfortunately, both of systems need to be further modified because of high background and low induction fold. In conclusion, I have successfully constructed Cu2+-specific WBCs utilizing the concept of an AND gate. Although optimization is necessary to enhance the detection performance of these two systems, they serve as a foundation for the future development of a more comprehensive detector.
Whole-cell biosensors (WBCs) utilize bacteria as hosts for detection. In order to detect environmental pollutants such as metal ions, I utilized genetic engineering techniques to introduce genes into bacteria to achieve our goal. WBCs are known for their low cost, simplicity, and environmentally friendly, which contribute to their rapid development. In my research, I employed C. metallidurans as the host organism and integrated the concept of an AND gate into plasmid design. I chose two promoters, PCopA (Cu2+, Zn2+, Cd2+) and PCopZ (Cu2+, Au3+, Ag+), to enhance the specificity of Cu2+ detection. Additionally, I introduced the recombinant tag Spy Tag/Catcher to improve the efficiency of complementation sfCherry3C(1-10), sfCherry3C(11), and fusion T7 RNA polymerase (C-T7/N-T7), serving as a signal amplifier to enhance Cu2+ detection performance. I successfully constructed Cu2+-specific strains in both of systems. The sfCherry3C system and T7 RNAP system exhibited good linear ranges of 2.5-250 μM and 0.1-5 μM, respectively. Despite their low limit of detection. Unfortunately, both of systems need to be further modified because of high background and low induction fold. In conclusion, I have successfully constructed Cu2+-specific WBCs utilizing the concept of an AND gate. Although optimization is necessary to enhance the detection performance of these two systems, they serve as a foundation for the future development of a more comprehensive detector.
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全細胞生物感測器, 重金屬, 調控系統, 邏輯閘, 重組蛋白, 耐金屬貪銅菌, 大腸桿菌, 銅離子, T7 RNA聚合酶, Whole-Cell Biosensors (WCBs), Regulatory Systems, Metals, Logic Gate, Recombinant Proteins, Cupriavidus metallidurans, E. coli, Copper ions