運用邏輯閘建構全細胞生物感測器視覺化檢測汞離子

Abstract

邏輯閘是基於布林邏輯運算的基礎元件，使用AND-gate系統進行設計的全細胞生物感測器結合多重輸入端進行調控，能有效提升全細胞生物感測器的靈敏性及專一性。本篇在大腸桿菌中研究三種邏輯閘元件，分別為調控型的HrpR/HrpS及重組型的Spy Tag/Spy Catcher與分裂的內含肽 (split intein)，成功在大腸桿菌內展現出符合AND-gate邏輯運算的能力，並詳細討論HrpR/HrpS系統及Spy Tag/Spy Catcher系統在汞離子檢測中的表現。透過比較不同的螢光報導基因：分裂的紅螢光蛋白 (sfCherry3C)及分裂的綠螢光蛋白 (sfGFP)，最終發現Spy Tag/Spy Catcher系統在重組sfGFP有顯著的效率，與單質體系統中以MerR基因組進行汞離子檢測有同等的螢光強度，並得到更低的偵測極限，低於臺灣對於工業排放廢水中汞的法規濃度。為了拓展汞離子檢測上的應用，本研究對調控蛋白MerR進行突變的定向進化，增進了對於汞離子的檢測能力，提升螢光強度以提升視覺化檢測的能力，最終可應用在真實水樣中檢測低濃度的汞離子；同時也開發出使用冷凍乾燥進行菌種的保存，能在室溫或是冰箱內進行儲存，探討冷凍乾燥粉末一個月內的保存能力，增加現場檢測的應用。The AND-gate system, capable of performing Boolean logic operations and incorporating multiple inputs for modulation, emerges as a promising avenue for enhancing sensitivity and specificity in whole-cell biosensors. This study investigates three types of logic gate elements in Escherichia coli: HrpR/HrpS, Spy Tag/Spy Catcher, and the split intein. It successfully demonstrates the capability of performing AND-gate logic operations in E. coli. The performance of the HrpR/HrpS system and the Spy Tag/Spy Catcher system in mercury ion detection is discussed in detail. By comparing different fluorescent reporter genes sfCherry3C and sfGFP, it was ultimately found that the Spy Tag/Spy Catcher system had significant efficiency in recombining sfGFP. This system achieved fluorescence intensity equivalent to the single-plasmid system using the MerR regulator gene for mercury ion detection. LOD is below Taiwan's regulatory concentration for mercury in industrial wastewater discharge. To expand the application of mercury ion detection, this study employed directed evolution of the regulatory protein MerR, enhancing its mercury ion detection capability and improving fluorescence intensity for visual detection. This allows for the detection of low concentrations of mercury ions in real water samples. Additionally, the study developed a method for preserving bacterial strains using freeze-drying, enabling storage at room temperature or in a refrigerator. The preservation capability of the freeze-dried bacterial powder was investigated over a month, increasing the applicability for on-site detection.