優化以邏輯閘(AND gate)建構之全細胞生物感測器並檢測苯乙胺

Abstract

苯乙胺是一種生物胺，同時也是一種中樞神經系統的興奮物質，通常存在於巧克力和發酵食品中，由於微生物中含有氨基酸脫羧基酶，因此檢測苯乙胺可得知食品的新鮮度。目前，科學家們使用高效液相色譜法、電化學法、螢光材料等儀器對苯乙胺進行檢測，我們實驗室則是使用全細胞生物感測器對苯乙胺進行定量。不過在先前的研究中，苯乙胺感測器並不專一，因此我們對以重組紅螢光蛋白及邏輯閘AND gate系統設計的全細胞生物感測器進行了報導基因、連接子、核醣體結合位點、啟動子的優化。另外，我們也藉由表達氧化酶 (TynA) 和苯乙醛脫氫酶 (FeaB) 蛋白使苯乙胺能夠快速轉化為苯乙醛及苯乙酸，增加檢測環境中苯乙醛和苯乙酸的濃度，使AND gate系統可以更快速的產生重組紅螢光訊號來增加感測器的專一性及優化誘導螢光結果，在研究過程中也找到了最佳的誘導時間和最專一的感測器，並對真實樣品進行初步的檢測，期望開發一個可以實際定量食品中的苯乙胺濃度的全細胞生物感測器。Phenylethylamine (PEA) is a biogenic amine and a central nervous system stimulant found in chocolate or fermented foods. Since microorganisms contain amino acid decarboxylase, the freshness of food can be determined by detecting phenethylamine. Currently, scientists use instruments such as HPLC, electrochemical methods, fluorescent materials, etc., to detect phenylethylamine. However, in previous studies from our laboratory, the whole-cell biosensors were not specific to phenethylamine. In this study, we optimized the reporter genes, linkers, ribosome binding sites, and promoters to construct a PEA-specific whole-cell biosensor. We also express the oxidase (TynA) and phenylacetaldehyde dehydrogenase (FeaB) proteins to convert phenylethylamine into phenylacetaldehyde and phenylacetic acid, increase the concentration of phenylacetaldehyde and phenylacetic acid in the environment. After that, AND gate system can generate recombinant red fluorescent proteins more quickly to increase the specificity of the sensor and optimize the induction fold. Furthermore, we determined the best induction time and the most specific sensor, and compared the results with real sample tests. This study has important implications for the convenience of phenethylamine sensing, and the development of this whole-cell biosensor can accurately quantify the concentration of phenylethylamine in food samples.