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研究生: 林青璇
Lin, Ching-Shiuan
論文名稱: 綠色螢光蛋白應用於大腸桿菌生物取像 系統之研究
The Study of Green Fluorescent Protein Applications for E.coli Bioimaging System
指導教授: 鄭智元
Jeng, Jr-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: 2006
畢業學年度: 94
語文別: 中文
論文頁數: 54
中文關鍵詞: 巨大原生質體綠色螢光蛋白
外文關鍵詞: GFP
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  •   本研究探討大腸桿菌綠色螢光蛋白基因(pGFP)在大腸桿菌Escherichia coli XL1-Blue 巨大原生質體之表現,確立以螢光顯微鏡及數位照相定量菌體內綠色螢光蛋白螢光強度之方法,並用此法來估算啟動(promoter)的效率。實驗發現pGFP 螢光基因可在大腸桿菌巨大原生質體表現,代表巨大原生質體具有正常細胞功能。比較空氣量對螢光亮度的影響,發現螢光強度會隨著空氣量的減少而延遲。本研究建立之數位影像系統可數值化螢光蛋白之螢光強度,具有快速、直接、簡便的優點。利用此結果,本研究之綠色螢光蛋白巨大原生質體之生物取像系統具有作為研究啟動子(promoter)效率及細胞微結構與生理反應工具的發展潛力。

      To develop a rapid reporter system for estimating the promoter efficiency in Escherichia coli XL1B, the expression pattern of the green fluorescent protein(GFP) in the giant protoplast was investigated. In addition, the methods for quantifying the fluorescent intensity of GFP by fluorescent microscopy as well as digital camera were illustrated. The results showed that the protoplasts of E. coli was able to express GFP, indicating that giant protoplasts possess cell functionality. The expression level of GFP in E. coli protoplasts was affected by the air level, i.e. the fluorescence intensity increases as the air level does. The digital imaging method with the advantages of rapidity, directness, and easiness established in the work could digitalize the intensity of fluorescence emitted by GFP. The results suggested that the tested plasmids were stable in recombinant cells, no matter cells in normal situation or becoming the giant protoplasts. In conclusion, the bioimaging system of the giant protoplast of E. coli could be a suitable method to estimate the promoter efficiency and promise to be a tool for studying the protein expression and cellular activity.

    中文摘要......................................................................I Abstract.....................................................................II 誌謝........................................................................III 圖目錄......................................................................VII 表目錄.......................................................................IX 符號表........................................................................X 第一章 緒論...................................................................1 1-1 前言......................................................................1 1-2 細菌細胞外部構造簡介......................................................1 1-2-1 細菌細胞外部構造........................................................1 1-2-2 革蘭氏陰性菌外部構造....................................................3 1-4 革蘭氏陰性菌原生質體形成方法..............................................6 1-5 生物冷光蛋白..............................................................8 1-6 綠色螢光蛋白..............................................................9 1-7 生物螢光影像.............................................................10 1-8 研究動機與目的...........................................................11 第二章 實驗材料與方法........................................................13 2-1 實驗材料.................................................................13 2-1-1 菌株...................................................................13 2-1-2 載體...................................................................13 2-1-3 藥品...................................................................14 2-1-4 培養基與Stock Solution ................................................15 2-1-5 實驗儀器...............................................................16 2-2 實驗方法.................................................................18 2-2-1 培養基配製.............................................................18 2-2-3 原生質體之製備與培養...................................................19 2-2-4 原生質體之螢光表現定量與圖像分析.......................................20 第三章 結果與討論............................................................21 3-1 菌株表現.................................................................21 3-1-1 菌株的螢光表現.........................................................21 3-1-2 E. coli XL1B‧pGFP 巨大原生質體與原始菌體之螢光表現....................21 3-2 螢光取像系統的建立.......................................................24 3-2-1 螢光亮區的選取.........................................................24 3-2-2 比螢光值及總螢光值的建立...............................................24 3-2-3 環境背景值的建立.......................................................25 3-2-4 質體螢光背景值的建立...................................................28 3-2-5 螢光灰度值的建立.......................................................28 3-3 E. coli XL1B 巨大原生質體之螢光表現......................................30 3-3-1 E. coli XL1B 巨大原生質體生長曲線......................................30 3-3-2 E. coli XL1B‧pGFP 巨大原生質體螢光表現................................31 3-3-3 以E. coli XL1B‧pGFP 之總螢光表現估算啟動子效率........................33 第四章 結論與未來展望........................................................48 參考文獻.....................................................................49 自述.........................................................................54

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