微生物金離子感測器與利用生物素酵素與其受體胜肽成像微生物中的蛋白質

Abstract

第一部分，我們運用基因工程技術設計出一組針對金離子有專一性且 選擇性良好的微生物感測器。本實驗是採用耐金屬貪銅菌 (Cupriavidus metallidurans)對金的調節基因組cup 系列的基因作為感測器的元件，並透 過基因工程技術以紅色螢光蛋白 (RFP)當作輸出訊號，用來檢測金屬。我 們將其應用於耐金屬貪銅菌 (Cupriavidus metallidurans) 和青枯桿菌 (Ralstonia eutropha)這兩種對環境適應力較強的土壤菌，一起探討這兩種微 生物感測器的專一性、耐受性和選擇性。由於傳統的儀器檢驗需要高成本、 高人力又耗時，可利用微生物感測器做簡單的操作，而且低成本又省時的 特性，在簡單的檢測及定量時，可以提供另一種替代的分析工具。 第二部分，我們運用生物素化作用 (biotinylation)標記法來觀察微生物 的蛋白質。螢光蛋白標記雖然是現在最普遍且高專一性的方法，但螢光蛋 白普遍都較大，會影響或干擾目標蛋白質的運動；生物素化作用標記則是 運用一段可結合生物素的胜肽 (biotin acceptor peptide, BAP)經過生物素的 酵素 (biotin ligase, BirA)催化，就可以與生物素 (biotin)結合，專一性高且 都是小分子，接下來就是比較兩個方法的效果。此系統原本應用於哺乳類 動物細胞的蛋白質研究，而本實驗將其技術應用於微生物的蛋白質標記， 不只是觀察單一的蛋白質，也可以觀察蛋白質與蛋白質之間的交互作用 (protein-protein interactions, PPIs)，拓展對於微生物的顯像技術和應用方法 的選擇。The part I of this thesis presents a fluorescence-based microbial sensor for the detection of heavy metal ions as a novel analytical tool for environmental applications. Our results demonstrate the effectiveness of whole-cell biosensor in the selective detection of gold ions. Cupriavidus metallidurans has a gold-resistance system controlled by cup regulon that has a gold-specific sensory protein CupR, a transcriptional regulator of MerR family. Two heavy-metal tolerant proteobacteria, Cupriavidus metallidurans and Ralstonia eutropha, were examined and showed great specificity. This work highlights the potential of employing engineered microbial strains as robust analytical tools. And, the part II describes a simple way to label proteins of microbes with biotinylation. The fusion of a fluorescent protein to target proteins has high specificity, but its size potentially disrupts trafficking and may cause misfolding. So we used an enzyme, biotin ligase (BirA) from E. coli, and a 15-amino acid peptide, biotin acceptor peptide (BAP), to achieve the high specificity and rapid labeling. Besides, BAP is a small tag and has less potential to disrupt target proteins. Microbial surface proteins tagged with BAP are biotinylated by biotin ligase. The other hand, this methodology also can label protein-protein interactions (PPIs) in cell. One protein of interest is fused to BirA, while another protein is fused to BAP substrate. Aside from mammalian cells, this work has demonstrated the adaptability of this technique to microbial system.