臺灣博碩士論文加值系統

　　本研究探討創傷弧菌藍色螢光蛋白基因之野生型(bfp)與突變型(bfp-D7)在大腸桿菌Escherichia coli XL1-Blue與創傷弧菌Vibrio vulnificus CKM-1巨大原生質體表現之差異性，並確立以螢光顯微鏡及數位照相定量菌體內創傷弧菌藍色螢光蛋白螢光強度之方法。實驗發現bfp與bfp-D7螢光基因皆可在大腸桿菌與創傷弧菌巨大原生質體表現，代表巨大原生質體具有細胞功能性，且bfp-D7螢光基因表現的螢光蛋白之螢光較bfp螢光基因產生者來得強。比較大腸桿菌原生質體與創傷弧菌原生質體，發現創傷弧菌原生質體的形成速度與死亡破裂速度均比大腸桿菌原生質體快，同時螢光基因表現程度亦較大腸桿菌高。創傷弧菌原生質體若培養溫度由30°C降至25°C明顯可延緩創傷弧菌原生質體凋亡時間，但亦會降低原生質體細胞生理活性。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。比較巨大原生質體及正常菌體之螢光強度，發現巨大原生質體螢光強度高於正常菌體且螢光維持時間較正常菌體長。此意味著基因重組菌在原生質體化及巨大化過程中質體不但沒有脫落，且產物之螢光蛋白濃度亦高於正常菌體。利用此結果，本研究之革蘭氏陰性菌巨大原生質體之生物取像系統具有作為研究細胞微結構與生理反應工具的發展潛力。

　The expression of the wild type (bfp) and mutation gene (bfp-D7) of blue fluorescent protein (BFP) isolated from Vibrio vulnificus CKM-1 in the giant protoplasts of Escherichia coli XL1B and V. vulnificus CKM-1 were investigated, respectively. In addition, the method for quantifying of the fluorescent intensity of BFP by fluorescent microscopy as well as digital camera was illustrated. The results first showed that both the protoplasts of E. coli and V. vulnificus were able to express bfp as well as bfp-D7, indicating that giant protoplasts possess cell functionality. The fluorescent intensity of the fluorescent protein expressed by bfp-D7 gene was stronger than that expressed by bfp gene. The growth and death rates of V. vulnificus protoplasts were higher than that of E. coli ones. As compared with E. coli, the expression level of BFP in V. vulnificus protoplasts was higher. The death rate of V. vulnificus protoplasts could be slow down at the expense of cellular activity when decreasing the incubation temperature from 30 to 25°C. The digital imaging method with the advantages of rapidity, directness, and easiness established in the work could digitalize the intensity of fluoresce emitted by BFP. The fluorescence produced by giant protoplasts is not only stronger but also longer than that emitted by normal cells. The result suggested that the plasmids were stable when the recombinant microbial cells becoming the giant protoplasts. Additionally, the yield of fluorescent protein produced by giant protoplasts was higher than normal cells. In conclusion, the bioimaging system of the giant protoplast of Gram-negative strains is promising to be a tool for studying the protein expression and cellular activity.

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...........................................XI

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......................2
1-3 原生質體簡介.................................8
1-4 革蘭氏陰性菌原生質體形成方法.................8
1-5 生物冷光蛋白................................10
1-6 創傷弧菌藍色螢光蛋白........................11
1-7 生物螢光影像................................12
1-8 研究動機與目的..............................13

第二章 實驗材料與方法............................15
2-1 實驗材料....................................15
2-1-1 菌株.....................................15
2-1-2 載體.....................................15
2-1-3 藥品.....................................16
2-1-4 培養基與 Stock Solution..................17
2-1-5 實驗儀器.................................18
2-2 實驗方法....................................20
2-2-1 培養基配製...............................20
2-2-2 菌種保存與活化...........................20
2-2-3 大腸桿菌之形質轉換.......................21
2-2-4 抽取少量質體DNA..........................22
2-2-5 DNA agarosegel分析.......................23
2-2-6 DNA agarosegel band純化..................23
2-2-7 電穿孔法.................................24
2-2-8 將bfp-D7構築於pUC19載體..................25
2-2-9 原生質體之製備與培養.....................26
2-2-10 原生質體之螢光表現定量與圖像分析........27

第三章 結果與討論................................28
3-1 載體構築與菌株表現..........................28
3-1-1 將D7基因構築於pUC19載體..................28
3-1-2 菌株的螢光表現...........................29
3-2 螢光取像系統的建立..........................34
3-2-1 螢光亮區的選取...........................34
3-2-2 螢光背景值的建立.........................35
3-2-3 螢光灰度值的建立.........................37
3-2-4 質體螢光背景值的建立.....................37
3-3 E. coli XL1B巨大原生質體之螢光表現..........40
3-3-1 E. coli XL1B巨大原生質體生長曲線.........40
3-3-2 E. coli XL1B host巨大原生質體螢光表現....41
3-3-3 E.coli XL1B•pUD7巨大原生質體螢光表現....41
3-3-4 E.coli XL1B•pUVF1巨大原生質體螢光表現...42
3-4 V. vulnificus CKM-1巨大原生質體之螢光表現...60
3-4-1 V. vulnificus CKM-1巨大原生質體生長曲線..60
3-4-2 V. vulnificus CKM-1host巨大原生質體螢光表現..............................................61
3-4-3 V. vulnificus CKM-1•pUD7巨大原生質體螢光表現........................................61
3-4-4 V. vulnificus CKM-1•pUVF1巨大原生質體螢光表現...........................................62
3-4-5 E. coli XL1B與 V. vulnificus CKM-1巨大原生質體螢光表現比較.................................62

第四章 結論與未來展望............................83
參考文獻.........................................85
自述.............................................88

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