葉怡均Yeh, Yi-Chun鄭文睿Cheng, Wen-Jui2022-06-089999-12-312022-06-082021https://etds.lib.ntnu.edu.tw/thesis/detail/4f2d49df3f193b2479452018f7203fd2/http://rportal.lib.ntnu.edu.tw/handle/20.500.12235/117219全細胞生物感測器是基於基因調控方式達成對特定重金屬、小分子達成感測，而又具有高靈敏、成本較低、生長需求較低等優點。本篇研究著重於使用基因工程優化全細胞生物感測器，並開發了金離子生物感測器，另一方面，則以邏輯閘概念初步開發了銅離子感測器與苯乙胺感測器。在金離子感測器方面，我們使用Cupriavidus metallidurans的轉錄蛋白CupR作為感測元件，利用CupR同源二聚體與金結合後，蛋白誘導啟動子DNA產生構型改變引起螢光蛋白表達，進而實現金離子感測，我們針對CupR蛋白結合序列、啟動子、核糖體結合位點、報導訊號等進行一系列優化。導入邏輯閘概念設計的兩種感測器，分別為：銅離子感測器、苯乙胺感測器，在銅離子感測器中，我們比較了leucine zipper與spy catcher/tag對於重組分裂的紅或綠色螢光的能力，另一方面，我們也使用了HrpR/S系統感測銅離子；在苯乙胺感測器方面，我們成功找出重組分裂紅色螢光蛋白的最佳化配對。Whole-cell biosensors are based on gene regulation methods to achieve sensing of specific heavy metals or small molecules, and they have the advantages of high sensitivity, low cost, and low growth requirements. This research focuses on the use of genetic engineering to optimize the whole-cell biosensor, including a gold ion biosensor, copper ion sensor, and the phenethylamine sensor.For the gold ion sensor, we use the CupR protein, a transcriptional protein in Cupriavidus metallidurans, as the sensing element. The gold(I)-dependent conformational change of CupR homodimers induces the activation of the promoter to express fluorescent protein achieving the detection of gold ions. We screened various CupR protein binding sequences, promoters, ribosome binding sites, reporting signals, etc. for the optimization of the purposed sensor. Two types of sensors using the logical gate concept (AND gate) design are: copper ion sensor and phenethylamine sensor. For the copper ion sensor, we compared the efficiency of leucine zipper and spy catcher/tag in terms of split-red/green fluorescent protein reassembly. In addition, we also used the HrpR/S system to detect copper ion. For the phenethylamine sensor, we figure out a best pair for split-red fluorescent protein reassembly.基因工程金離子銅離子重金屬耐金屬貪銅菌CupR操縱子全細胞生物感測器邏輯閘苯乙胺重組螢光蛋白Genetic engineeringGold ionsCopper ionsHeavy metalCupriavidus metalliduransCupR regulonWhole-cell biosensorLogic gatePhenylethylamine (PEA)Recombinant fluorescent protein學術論文