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研究生：	蔡東育 Tsai, Dong-Yu
論文名稱：	細菌表面顯示金屬結合胜肽作為對硝基苯胺還原反應之生物催化劑與發展貴重金屬離子的 FRET 生物感測器 Bacterial surface display of metal binding peptides as whole-cell biocatalysts for 4-nitroaniline reduction and a FRET-based biosensor for detection of noble metal ions
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：	2016
畢業學年度：	104
語文別：	中文
論文頁數：	91
中文關鍵詞：	表面顯示技術、金和鉑奈米粒子、生物催化劑、對硝基苯胺還原反應、螢光共振能量轉移、青色螢光蛋白、黃色螢光蛋白、CueR 轉錄調節蛋白
英文關鍵詞：	surface display, IgA protease, gold and platinum nanoparticles, whole-cell biocatalyst, 4-nitroaniline reduction, copper transcriptional regulator CueR
DOI URL：	https://doi.org/10.6345/NTNU202203830
論文種類：	學術論文
相關次數：	點閱：157 下載：5
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利用表面顯示技術以源自淋病奈瑟菌 (Neisseria gonorrheaea) 的IgA protease 作為攜帶蛋白，將金屬結合胜肽表現於Escherichia coli和Ralstonia eutropha的外膜上，回收與合成金和鉑奈米粒子。研究結果顯示，膜外表現金屬結合胜肽的細菌不僅能夠還原金和鉑離子，還能將奈米粒子透過金屬結合胜肽表現在細菌表面上。接著我們以吸附金屬奈米粒子的細菌作為生物催化劑，成功地催化對硝基苯胺的還原反應。此外，我們以蛋白質工程的方式設計出對一價銅、一價銀和三價金離子具有選擇性的螢光共振能量轉移蛋白質感測器。以感測蛋白A-66 (CFP-GGSGGS-CueR-GGSGGS-YFP)，進行試管內與活體細胞之金屬離子監測應用。

A surface display system was used for green synthesis of gold and platinum nanoparticles via an autotranspoter protein, immunoglobulin A protease (IgA protease) from Neisseria gonorrheae as carrier proteins. Metal binding peptides were expressed on the outer membranes of Escherichia coli and Ralstonia eutropha to recover metal ions. The results of this study showed that cells expressing metal binding peptides could synthesize and display the nanoparticles on the cell surfaces, simultaneously. Next, the metal nanoparticle-coated microorganisms were used as whole-cell biocatalysts for the reduction of 4-nitroaniline. Besides, we designed a protein-based fluorescence resonance energy transfer (FRET) reporter for detection of copper (I), silver (I), and gold (III) ions. The A-66 reporter (CFP-GGSGGS-CueR-GGSGGS-YFP) was selected for in vitro and in vivo metal ions detection.

誌謝    i
中文摘要    ii
Abstract    iii
Contents    iv
List of Figures    vii
List of Tables    xiii
Part I    14
Chapter 1    Introduction    15
1.1    金屬奈米材料的催化    15
1.2    金屬奈米粒子生物合成    17
1.3    微生物細胞表面顯示技術    19
1.4    研究動機與目的    22
Chapter 2    Materials and Experimental Methods    23
2.1    實驗儀器    23
2.2    實驗藥品    24
2.3    實驗方法    25
2.3.1    質體設計    25
2.3.2    利用螢光顯微鏡分析紅色螢光蛋白的位置    28
2.3.3    金奈米粒子的合成    28
2.3.4    比較多種表面顯示系統    28
2.3.5    金與鉑奈米粒子生物合成    29
2.3.6    對硝基苯胺的還原反應    29
Chapter 3    Results and Discussions    31
3.1    建構微生物表面顯示系統    31
3.2    比較表面顯示系統之成效    33
3.3    利用微生物合成金屬奈米粒子    35
3.4    全細胞 (whole-cell) 生物催化對硝基苯胺的還原反應    38
Chapter 4    Conclusions    44
Part II    45
Chapter 1    Introduction    46
1.1    微生物金屬調控    46
1.2    MerR-type的銅離子調節蛋白 (transcriptional regulator)    47
1.3    螢光蛋白之應用    49
1.4    研究動機與目標    51
Chapter 2    Materials and Experimental Methods    53
2.1    實驗儀器    53
2.2    實驗藥品    54
2.3    實驗方法    57
2.3.1    螢光蛋白感測器的設計    57
2.3.2    螢光蛋白感測器純化 (protein purification)    59
2.3.3    十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (Sodium dodecyl sulfate polyacrylamide gel electrophore, SDS-PAGE)    59
2.3.4    螢光蛋白感測器濃度測定 (Bradford protein assay)    60
2.3.5    篩選螢光蛋白與CueR (S) 間不同鏈長設計的螢光蛋白感測器    60
2.3.6    金屬選擇性 (selectivity)    61
2.3.7    金屬靈敏性 (Sensitivity)    61
2.3.8    金屬干擾性 (Interference)    61
2.3.9    細菌體內偵測實驗 (in vivo)    62
Chapter 3    Results and Discussion    63
3.1    篩選以螢光共振能量轉移所建立的 (FRET-based) 螢光蛋白感測器    63
3.2    螢光蛋白感測器選擇性探討    65
3.3    螢光蛋白感測器其螢光強度與金、銀、銅離子濃度關係    66
3.4    螢光蛋白感測時金屬離子間的干擾性    68
3.5    螢光蛋白感測器在E. coli中對銅離子的感測情形    69
Chapter 4    Conclusions    71
Reference    72
Appendix A    Primers, Plasmids and Strains    74
Appendix B    EDS spectra    88
                                

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