透過基因工程優化全細胞生物感測器：主題一、以CupR蛋白開發的金離子感測器 主題二、以TyrR蛋白開發的酪胺酸感測器

Genetic Engineering to Optimize Whole-cell Biosensor: Part I. CupR-based Sensor for Au(III) Detection. Part II. TyrR-based Sensor for Tyrosine Detection.

張謙獻(Zhang Qian-Xian)

指導教授 ： 葉怡均

國立臺灣師範大學/理學院/化學系/碩士(2019年)

https://doi.org/10.6345/NTNU201900430

摘要

全細胞生物感測器在感測小分子化合物時具有極大的優勢，能夠使用簡單且低成本的裝置達成原本需要利用貴重儀器才能達到的靈敏度，並且有專一強、對培養環境需求低等優點。在本研究中主要使用基因工程優化全細胞生物感測器，使其能夠分別對金離子及酪胺酸進行感測。在金離子感測器的方面，我們運用Cupriavidus metallidurans中的CupR蛋白作為調節機制，使用專一性蛋白結合序列對報導的螢光基因進行表現。藉由運用同屬MerR家族但不同蛋白的專一性蛋白結合序列，探討其在細胞內與蛋白結合能力。對於優化 TyrR 作為調控蛋白的酪胺酸全細胞生物感測器，使用了細菌代謝芳香族胺基酸的重要基因組TyrR 調控組作為調控機制，利用了兩個分別響應培養物中不同酪胺酸濃度的啟動子，控制紅色及綠色螢光信號的輸出。最後開發了一種無須額外添加其餘試劑的量測平台，讓使用者只需要加入樣品，就可藉由量測數據得知樣品中酪胺酸濃度。

關鍵字

基因工程 ； 金離子 ； 耐金屬貪銅菌 ； CupR操縱子 ； 酪胺酸 ； TyrR調控組 ； 全細胞生物感測器

並列摘要

The whole-cell biosensor has great advantages in sensing small molecule compounds. It can achieve sensitivity through a simple setup and low-cost device instead of using expensive instruments. Furthermore, it has the advantages of great specificity and low demand for the cultivated environment. In this research, genetic engineering was used to optimize the whole-cell biosensor to enable sensing of gold ions and tyrosine, respectively. For gold ion sensors, we used the CupR protein, which was adopted from Cupriavidus metallidurans, as our regulatory protein mechanism to express the fluorescent proteins. To investigate the binding ability between the protein binding sequences and CupR in the cells, we changed the CupR binding sequences of the promoters. For the development of tyrosine whole-cell biosensor, we used TyrR as a regulatory protein. TyrR regulator plays an important role in bacteria for the metabolization of aromatic amino acids. Two tyrosine-responsive promoters are used to differentially control the production of red and green fluorescent proteins signals in response to tyrosine levels in culture. To make the device more user-friendly, we developed a reagentless platform in which biocompatible agarose is used as an entrapment agent with cell sensors and growth media mounted within the gel matrix.

並列關鍵字

Genetic engineering ； Gold ions ； Cupriavidus metallidurans ； CupR operon ； Tyrosine ； TyrR regulon ； Whole-cell biosensor

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