以邏輯閘建構全細胞生物感測器檢測重金屬離子

Abstract

全細胞生物傳感器是一種將活細胞作為生物識別元件的生物傳感器，可用於檢測特定有毒物質或代謝物等，具有對環境友善、低成本和可攜帶性等優點。本論文主要基於邏輯閘概念去設計出不同的全細胞生物感測器：(1)在大腸桿菌中比較不同合成基因的汞離子感測器；(2)在耐金屬貪銅菌中開發銅金離子感測器。第一部分，根據邏輯閘概念，我們分別設計四種經過重組基因修飾的全細胞生物傳感器。在汞離子(Hg2+)和異丙基β-d-1-硫代半乳糖苷(IPTG)分子的控制下，分別比較了四對基因，包括LuxRI、HrpRS、Spy Tag/Catcher和E4/K4捲曲螺旋。此外，我們還研究了這些全細胞生物傳感器在各種pH水平下及其金屬干擾。第二部分，根據第一部份結果我們選擇成功表達的三個系統(LuxRI、HrpRS、Spy Tag/Catcher)，移到耐金屬貪銅菌(Cupriavidus metallidurans)中建立基於邏輯閘概念之銅及金離子感測器。Whole-cell biosensors are a type of biosensor that incorporates living cells as a biorecognition element, which can be used to detect specific toxic substances or metabolites. They have many advantages of being environmentally friendly, low-cost, and portable. This thesis mainly designs different whole-cell biosensors based on the concept of the logic gate: (1) Comparing mercury whole-cell biosensors combined with different synthetic genes in Escherichia coli; (2) Developing copper and gold ions whole-cell biosensors in Cupriavidus metallidurans.In the first part, according to the logic gate concept, we designed four whole-cell biosensors that were modified by recombinant genes, separately. Four pairs of genes were compared respectively including LuxRI, HrpRS, Spy Tag/Catcher, and E4/K4 coiled-coil under the control of mercury ions (Hg2+) and isopropyl β-d-1-thiogalactopyranoside (IPTG) molecules. Moreover, we also investigated these whole-cell biosensors under various pH levels and their metal interference.In the second part, according to the results of the first part, we selected three systems (LuxR/LuxI, HrpR/HrpRS, Spy Tag/Catcher) optimized from the first part, and expressed them in Cupriavidus metallidurans to detect copper and gold ions based on the logic gate concept.