| 研究生: |
李瑞俞 Li, Ruei-Yu |
|---|---|
| 論文名稱: |
以生物取像系統探討在不同培養條件下之基因重組大腸桿菌內涵體的形成 Study of Inclusion Body Formation under Various Culture Conditions in Recombinant Escherichia coli with 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: | 2007 |
| 畢業學年度: | 95 |
| 語文別: | 中文 |
| 論文頁數: | 65 |
| 中文關鍵詞: | 生物取像系統 、原生質體 、內涵體 |
| 外文關鍵詞: | bioimaging system, protoplast, inclusion body |
| 相關次數: | 點閱:126 下載:1 |
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生物取像系統(bioimaging system)使用螢光蛋白當作報導蛋白,ㄧ般應用於真核生物,可即時精確地觀察到螢光蛋白在細胞或組織之形成及分布狀態;而原核生物細胞體積只有真核生物細胞的千分之ㄧ,通常無法使用生物取像系統精確地觀察螢光蛋白在胞內之分布狀態。本研究以特殊培養方法,將基因重組大腸桿菌BL21(DE3)/pET-D7做成與酵母菌同等大小之巨大原生質體(giant protoplast)。在IPTG 誘導下,由於藍色螢光蛋白-D7 的大量表現,可即時觀察到內涵體之形成,藉此可評估基因重組大腸桿菌形成內涵體之培養條件。
實驗發現在不同培養條件下,藉由螢光顯微鏡觀察巨大原生質體螢光表現差異,可得到隨著誘導劑及培養溫度的增加或培養液pH 值的下降,其內涵體的形成有增加的趨勢。
The bioimaging system usually utilizes fluorescence protein as a reporter gene in eukaryotic systems, and it gives us an easy way to real-time monitor the distribution of fluorescence protein in cells. Eukaryote is thousand times the size of prokaryote, so it is not suitable to this system. In our research, giant protoplasts with size similar to Saccharomycete were prepared from recombinant Escherichia coli BL21(DE3)/pET-D7. The expression of D7 can be induced by IPTG to monitor inclusion body formation in real time. Using this approach, we can study inclusion body formation in recombinant Escherichia coli under various culture conditions.
By the fluorescence microscope, we monitored the expression of fluorescence in the giant protoplast. The difference in fluorescence under various culture contitions revealed that inclusion body formation would
increase with increasing IPTG, with increasing induction temperature or with decreasing pH value.
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