開發以轉錄因子調控的全細胞生物感測器: 主題一、利用耐金屬貪銅菌偵測銅離子 主題二、藉由雙重訊號輸出同時偵測並定量苯乙胺與苯乙酸

Abstract

銅離子在人體內扮演著重要的角色，例如參與呼吸作用電子傳遞鏈以及一些神經訊息的傳遞。然而銅離子濃度過高也會導致一些疾病，例如威爾森氏症、帕金森氏症。苯乙胺是神經傳導物質，苯乙酸是其代謝產物，一些疾病如苯丙酮尿症和精神分裂症患者的尿液中可偵測到過量的苯乙胺和苯乙酸。本研究期望能提供以微生物系統作為感測的新方法。偵測銅離子方面我們表達耐金屬貪銅菌金離子調控組CueR的啟動子PcopZ和銅離子調控組CopSR的啟動子PcopA，偵測苯乙胺和苯乙酸方面同時表達大腸桿菌的單胺類調控組Mao、苯乙酸代謝調控組Paa，兩者分別接上紅色螢光蛋白與綠色螢光蛋白作為雙重訊號輸出。藉由檢測紅色螢光與綠色螢光之訊號，我們設計的感測器對銅離子、苯乙胺、苯乙酸的偵測具有高度專一性和靈敏性。

Copper ions act as an important role in our body, involving cellular respiration and signal transduction. Some neurodegenerative diseases, such as Wilson diseases and Alzheimer’s disease, are resulted from high copper ion concentration. β-Phenylethylamine (PEA) is an important neurotransmitter in our body and phenylacetic acid (PA) is its metabolite. Patients with special diseases such as phenylketonuria and paranoid schizophrenia have high concentration of PEA and PA in body fluids. We constructed two dual signal whole-cell biosensors with the expression GFP and RFP for detection copper ions, PEA, and PA. First, we used promoter PcopZ and PcopA in Cupriavidus metallidurans to exclude the interference caused by other metal ions. Second, we used PEA and PA degradation operon in Escherichia coli to detect PEA and PA simultaneously. By monitoring RFP and GFP fluorescence, we both had good sensitivity and selectivity for detection of Cu2+, PEA, and PA.