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研究生: 游象淳
You, Siang-chun
論文名稱: 創傷弧菌藍色螢光蛋白在基因重組大腸桿菌生物取像系統應用之研究
The Imaging Analysis of Recombinant Escherichia coli Using The Blue Fluorescence Protein from Vibrio vulnificus
指導教授: 鄭智元
Cheng, Chu-Yuan
學位類別: 碩士
Master
系所名稱: 工學院 - 化學工程學系
Department of Chemical Engineering
論文出版年: 2008
畢業學年度: 96
語文別: 中文
論文頁數: 68
中文關鍵詞: 藍色螢光蛋白
外文關鍵詞: Blue Fluorescence Protein
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  • 海洋創傷弧菌之藍色螢光蛋白與綠色螢光蛋白相同,可當作報導蛋白使用,惟至目前關於藍色螢光蛋白螢光特性研究之報告仍不多見。
    本研究主要目的為探討藍色螢光蛋白之螢光蛋白濃度與螢光強度之關聯,並依據此結果將巨大原生質體在螢光顯微鏡下所觀察之螢光強度轉換成所生產之螢光蛋白濃度,以提升巨大原生質體在生物取像系統之泛用性。

    Both the blue fluorescence protein (BFP) and green fluorescence protein (GFP) isolated from Vibrio vulnificus could be utilized as reporter proteins. However, unlike GFP, very few studies on BFP have been published to date.
    The correlation between the fluorescent intensity and the BFP concentration was examined in this study. After purifying the BFP expressed by Escherichia coli BL21(DE3)/pET-D7, the fluorescence emitted by various concentrations of BFP were correlated. This correlation could transform the emitted fluorescence observed by a fluorescence microscope into BFP concentration, which might enlarge the applicability of E. coli giant protoplasts in a bioimaging system.

    目錄 中文摘要………………………………………………………... I 英文摘要………………………………………………………... II 誌謝……………………………………………………………... III 目錄……………………………………………………………... IV 表目錄…………………………………………………………... VII 圖目錄…………………………………………………………... VIII 符號表…………………………………………………………... X 第一章 緒論………………………………… ………… 1 1-1 前言………………………………………………………… 1 1-2 創傷弧菌(Vibrio vulnificus)…………………………. 2 1-3 細菌細胞外部構造簡介…………………………………… 3 1-3-1 細菌細胞外部構造…………………………………. 3 1-3-2 革蘭氏陰性菌外部構造……………………………. 8 1-4 原生質體的簡介…………………………………………… 13 1-5 革蘭氏陰性菌原生質體形成方法………………………… 14 1-6 生物冷光蛋白……………………………………………… 16 1-7 創傷弧菌(Vibrio vulnificus)藍色螢光蛋白………….. 19 1-8 報導基因(reporter gene)……………………………… 20 1-9 重組基因的誘導…………………………………………… 22 1-10 生物螢光影像…………………………………………….. 23 1-11 研究動機與目的………………………………………….. 24 第二章 實驗材料與方法………………………… …… 25 2-1 實驗材料…………………………………………………… 25 2-1-1 藥品…………………………………………………. 25 2-1-2 實驗儀器……………………………………………. 26 2-1-3 菌株…………………………………………………. 28 2-1-4 培養基、緩衝液與Stock Solution………………… 28 2-2 實驗方法…………………………………………………… 32 2-2-1 菌株保存與活化……………………………………. 32 2-2-2 原生質體之製備與培養……………………………. 32 2-2-3 螢光蛋白之純化……………………………………. 34 2-2-4 蛋白質定量…………………………………………. 36 2-3 分析方法…………………………………………………… 36 第三章 結果與討論…………………………… ……… 37 3-1 菌株在固態培養基上的螢光表現………………………… 37 3-2 菌體的生長………………………………………………… 39 3-2-1 原始菌之生長曲線及檢量線…………………..... 39 3-2-2 巨大原生質體之生長曲線…………………………. 39 3-3 藍色螢光蛋白純化及其螢光強度分析…………………… 42 3-3-1 藍色螢光蛋白純化原理……………………………. 42 3-3-2 電泳之比對分析……………………………………. 42 3-3-3 藍色螢光蛋白溶液的濃度標定……………………. 43 3-3-4 藍色螢光蛋白的螢光強度…………………………. 44 3-3-5 藍色螢光蛋白與濃度之檢量線製定………………. 51 3-4 巨大原生質體中藍色螢光蛋白的測定與分析…………… 55 3-4-1 巨大原生質體螢光亮區選取與螢光強度分析……. 55 3-4-2 巨大原生質體OD600值對顯微鏡下螢光強度關係.. 58 3-4-3 以螢光光度計分析巨大原生質體的螢光強度……. 59 3-4-4 螢光光度計與螢光顯微鏡分析方法之整合………. 61 第四章 結論………………………………… ………… 62 參考文獻………………………………………………………... 64 表目錄 表1-8-1 Reporter gene………………………………………… 21 表2-1-1 LB培養基組成……………………………………… 28 表2-1-2 GP培養基組成……………………………………… 29 表2-1-3 SP buffer組成……………………………………….. 29 表2-1-4 Phosphate buffer組成……………………………... 30 表2-1-5 Strip buffer組成…………………………………... 30 表2-1-6 Charge buffer組成………………………………... 30 表2-1-7 Elute buffer組成………………………………….. 30 表2-1-8 Stock solution……………………………………... 31 表2-2-1 蛋白純化程序濃度操作表………………………….. 35 圖目錄 圖1-3-1 膜構造……………………………………………….. 6 圖1-3-2 革蘭氏陽性菌與革蘭氏陰性菌細胞壁構造之區別.. 7 圖1-3-3 細菌細胞外部構造………………………………….. 11 圖1-3-4 Peptidoglycan之結構……………………………... 12 圖1-5-1 原生質體形成步驟圖……………………………….. 15 圖1-6-1 激發電子能階圖…………………………………….. 18 圖1-6-2 冷光細菌細胞藉由luciferase在氧氣存在發光機制. 18 圖1-9-1 基因的調控中誘導型調節系統…………………….. 22 圖3-1-1 菌株在LB固態培養基受到UV光照射前後的照片. 38 圖3-2-1 原始菌誘導後的生長曲線………………………….. 40 圖3-2-2 吸光值對乾菌重之檢量線………………………….. 40 圖3-2-3 巨大原生質體誘導後的生長曲線………………….. 41 圖3-3-1 電泳分析圖………………………………………….. 43 圖3-3-2 可見光光譜範圍…………………………………….. 44 圖3-3-3 BFP螢光吸收光譜………………………………….. 45 圖3-3-4 BFP螢光吸收光譜………………………………….. 46 圖3-3-5 BFP螢光吸收光譜………………………………….. 46 圖3-3-6 BFP激發光吸收光譜……………………………….. 47 圖3-3-7 NADPH螢光吸收光譜……………………………… 49 圖3-3-8 BFP螢光吸收光譜………………………………….. 49 圖3-3-9 BFP + NADPH螢光吸收光譜………………………. 50 圖3-3-10 BFP螢光吸收光譜……………………............. 52 圖3-3-11 BFP螢光吸收光譜…………………………………. 52 圖3-3-12 BFP螢光吸收光譜……………………………….... 53 圖3-3-13 BFP螢光吸收光譜………………………………… 53 圖3-3-14 BFP濃度與螢光強度之關係圖…………………… 54 圖3-4-1 巨大原生質體照片………………………………….. 56 圖3-4-2 使用IPP軟體擷取螢光亮區圖……………………... 57 圖3-4-3 使用IPP軟體分析藍色光亮度……………………... 57 圖3-4-4 巨大原生質體OD600值與總螢光強度關係圖……… 58 圖3-3-5 巨大原生質體中BFP螢光吸收光譜………………. 59 圖3-4-6 巨大原生質體OD600值與螢光強度關係圖………… 60 圖3-4-7 螢光顯微鏡與螢光光度計分析法之關係圖……….. 61

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