以邏輯閘及緩衝終結子強化苯乙胺全細胞生物感測器並發展苯乙胺生物感測器的延伸應用

Abstract

苯乙胺為一種神經傳導物質於腦中扮演重要的角色，苯乙胺又為許多藥物、神經傳導物質的基本單元，因此顯現檢測苯乙胺的重要性。先前研究中，透過在大腸桿菌邏輯閘系統來提高傳感器對PEA的特異性。然而PtynA啟動子的洩漏表達問題導致苯乙酸對傳感器的干擾，因此在本篇的研究中，測試不同的終止子以解決啟動子洩漏表達問題。最終PEA生物傳感器的動態範圍為1 µM到400 µM，線性範圍在50 µM到400 µM之間，偵測極限到達1.86 µM。另外也發展了水凝膠封裝細菌的系統，優化了兩種水凝膠：海藻酸－丙烯醯胺、海藻酸－聚合離胺酸水凝膠，針對培養條件、製備條件進行優化並發現使用0.25倍M9培養基製備的海藻酸－聚合離胺酸水凝膠在1倍的M9培養基下培養16小時能夠得到最好的結果，同時我們也將最優化菌株YCY_1644封裝進水凝膠可以看到與空白實驗明顯差別的螢光結果，開發出可以用於實驗室所有生物感測器的水凝膠，以達到全細胞生物感測器臨場檢測以及可攜性的價值。此外我們針對大腸桿菌MG1655染色體中對於苯乙胺調控的相關基因feaR、feaB以及tynA進行基因剔除，並發展螢光探針para-methoxy-2-amino benzamidoxime ( PMA )期許未來能夠進行安非他命以及篩選TynA蛋白突變體之應用。Phenylethylamine (PEA) is a neurotransmitter that plays an important role in the brain and serves as a basic unit for many drugs and neurotransmitters, highlighting the significance of detecting PEA. Previous research enhanced the specificity of PEA sensors using a logic gate system in Escherichia coli. However, the issue of promoter leakage from the PtynA led to interference from phenylacetic acid on the sensor. Therefore, this study tested different terminators to address the problem of promoter leakage. Ultimately, the dynamic range of the PEA biosensor was 1 µM to 400 µM, with a linear range between 50 µM and 400 µM and Limit of Detection ( LOD ) reaching 1.86 µM. Additionally, a system for encapsulating bacteria in hydrogels was developed, optimizing two types of hydrogels: alginate-acrylamide and alginate-poly-lysine hydrogels. Optimization of culture and preparation conditions revealed that using alginate-poly-lysine hydrogel prepared with 0.25x M9 medium and cultured in 1x M9 medium for 16 hours yielded the best results. Furthermore, encapsulating the optimized strain YCY_1644 in the hydrogel produced significant fluorescent results compared to blank experiments. This development of a hydrogel suitable for all laboratory biosensors aims to achieve on-site detection and portability for whole-cell biosensors. Additionally, genes related to PEA regulation in the Escherichia coli MG1655 chromosome, including feaR, feaB, and tynA, were deleted, and a fluorescent probe, para-methoxy-2-amino benzamidoxime (PMA), was developed, aiming for future applications in amphetamine detection and TynA protein mutant screening