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研究生：	曾學瑋 Tseng, Hsueh-Wei
論文名稱：	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 Microbial Sensor for the Detection of Gold and Imaging Microbial Proteins with Biotin Ligase and Biotin Acceptor Peptide
指導教授：	葉怡均 Yeh, Yi-Chun
學位類別：	碩士 Master
系所名稱：	化學系 Department of Chemistry
論文出版年：	2014
畢業學年度：	102
語文別：	中文
論文頁數：	108
中文關鍵詞：	微生物感測器、耐金屬貪銅菌、金的調節基因組cup 、青枯桿菌、生物素化作用標記法、可結合生物素的胜肽、生物素的酵素、生物素
英文關鍵詞：	fluorescence-based microbial sensor, Cupriavidus metallidurans, gold-resistance system controlled by cup regulon, Ralstonia eutropha, biotinylation, biotin acceptor peptide (BAP), biotin ligase (BirA), biotin
論文種類：	學術論文
相關次數：	點閱：112 下載：3
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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.

目錄 ............................................................................................................. I
圖表目錄 .................................................................................................... V
摘要 .......................................................................................................... IX
Abstract ..................................................................................................... X
I. 微生物重金屬感測器 ......................................................................... 1
第一章 緒論............................................................................................... 1
1.1 生物感測器 (biosensor) .......................................................................... 1
1.2 微生物與重金屬 ...................................................................................... 2
1.2.1 生物礦化作用 (biomineralization) ............................................................. 2
1.2.2 微生物對金屬的耐受性 ............................................................................. 2
1.2.3 MerR 家族的轉錄調節因子 (transcriptional regulator of MerR family) ... 4
1.3 金金屬微生物感測器 (microbial sensor of gold) .................................. 6
1.4 微生物宿主 .............................................................................................. 7
1.5 研究動機 .................................................................................................. 9
1.6 研究目標 ................................................................................................ 10
第二章 實驗藥品與器材 ........................................................................ 11
2.1 實驗儀器 ................................................................................................ 11
2.2 實驗藥品 ................................................................................................ 12
第三章 實驗方法 .................................................................................... 14
II
3.1 微生物感測器的設計 ............................................................................ 14
3.1.1 質體設計 ................................................................................................... 14
3.1.2 質體基因工程 (cloning) ........................................................................... 15
3.2 實驗步驟 ................................................................................................ 21
3.2.1 專一性 (specificity) .................................................................................. 21
3.2.2 干擾性 (interference) ................................................................................ 22
3.2.3 耐受性 (tolerance) .................................................................................... 22
3.2.4 靈敏度 (sensitivity) .................................................................................. 23
3.2.5 最低偵測極限 (limit of detection, LOD) ................................................. 23
第四章 實驗結果與討論 ........................................................................ 24
4.1 微生物金離子感測器之篩選 ................................................................ 24
4.1.1 質體PcupA/R-cupR-rfp 和宿主組合之金離子篩選 ................................ 24
4.1.2 質體cupR(rev)-PcupA/R-PcupC-rfp 和宿主組合之金離子篩選 ............ 33
4.2 微生物金離子感測器中CupR 調控蛋白對金離子的感測 ................ 42
4.3 微生物金離子感測器之重金屬環境干擾及選擇性 ............................ 45
4.3.1 質體cupR(rev)-PcupA/R-PcupC-rfp 和C. metallidurans 宿主組合之微生
物金離子感測器對其他重金屬的干擾探討 ..................................................... 45
4.3.2 質體cupR(rev)-PcupA/R-PcupC-rfp 和R. eutropha 宿主組合之微生物金
離子感測器對其他重金屬的干擾探討 ............................................................. 48
4.4 微生物金離子感測器之螢光強度與金離子濃度關係 ........................ 52
4.4.1 質體PcupA/R-cupR-rfp 和C. metallidurans 宿主組合之金離子濃度探討
............................................................................................................................. 52
4.4.2 質體cupR(rev)-PcupA/R-PcupC-rfp 和C. metallidurans 和R. eutropha 宿
主組合之金離子濃度探討 ................................................................................. 53
4.5 微生物R. eutropha 金離子感測器改良 ............................................... 61
III
4.5.1 質體cupR(rev)-PcupA/R-PcupC-rfp-cupC 和R. eutropha 宿主之組合 . 61
4.5.2 質體加入cupA 和cupC 的不同組合並與R. eutropha 宿主組成感測器
............................................................................................................................. 63
4.6 螢光顯微鏡下的微生物金離子感測器效率 ........................................ 67
第五章 結論............................................................................................. 69
II. 微生物蛋白質顯像技術 ................................................................ 71
第六章 緒論............................................................................................. 71
6.1 生物顯像技術 (biological imaging) ..................................................... 71
6.2 生物素化作用 (biotinylation) ............................................................... 73
6.3 目標蛋白質 (target proteins) ................................................................ 74
6.3.1 細胞表面的目標蛋白質 ........................................................................... 74
6.3.2 蛋白質與蛋白質之間的作用 (Protein-protein interaction) .................... 77
6.4 研究動機 ................................................................................................ 80
6.5 研究目標 ................................................................................................ 81
第七章 實驗藥品與器材 ........................................................................ 82
7.1 實驗儀器 ................................................................................................ 82
7.2 實驗藥品 ................................................................................................ 83
第八章 實驗方法 .................................................................................... 86
8.1 質體設計和基因工程 (cloning) ........................................................... 86
8.1.1 質體設計 ................................................................................................... 86
8.1.2 質體基因工程 (cloning) ........................................................................... 87
8.2 蛋白質純化 (protein purification) ........................................................ 91
IV
8.2.1 蛋白質標記 (His-Tag) .............................................................................. 91
8.2.2 十二烷基硫酸鈉聚丙烯酰胺凝膠電泳 (sodium dodecyl sulfate
polyacrylamide gel electrophore, SDS-PAGE) .................................................... 93
8.2.3 蛋白質純化 ............................................................................................... 96
8.2.4 蛋白質濃度測定 (Bradford protein assay) .............................................. 97
8.2.5 鎳樹脂回收 (recovery of Ni-NTA resin) .................................................. 98
8.3 微生物蛋白質標記 (microbial proteins labeling) ................................ 99
第九章 實驗結果與討論 ...................................................................... 101
9.1 微生物外膜蛋白質標記 ...................................................................... 101
9.2 微生物體內蛋白質與蛋白質間的交互作用觀察 .............................. 103
第十章 結論........................................................................................... 106
參考文獻 ..................................................................................................... 107
                                

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