臺灣博碩士論文加值系統

生物取象系統使用使用螢光蛋白當作報導蛋白，一般應用於真核生物，可即時精確地觀察到螢光蛋白在細胞或組織之行程及分布狀態；而原核生物細胞體積只有真核生物細胞的千分之一，通常無法使用生物取象系統精確地觀察螢光蛋白在細胞內之分佈狀態。本研究以特殊培養方法，將基因重組大腸桿菌BL21(DE3)/ pET21a-GFP 做成與酵母菌同等大小之巨大原生質體(giant protoplast) 。在IPTG誘導下，由於綠色螢光蛋白的大量表現，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。

Bioimaging system usually utilize fluorescence protein as a reporter gene in eukaryotic system, and it gives a easy way for us to real-time monitor the distribution of fluorescence protein in bacteria or cell.Eukaryote is thousand times the size of prokaryote, so it is not suitable to this system.In our reserach, recombinant Escherichia coli BL21(DE3)/ pET21a-GFP is used to prepare as giant protoplast which size is similar to saccharomycete with particular method. The expression of GFP can be induced by IPTG to real-time monitor inclusion body formation.Using this approach, we can study inclusion body formation under various culture conditions in recombinant Escherichia coli.
By monitoring the fluorescence protein formation in the giant protoplast under various culture conditions, the result indicated that inclusion body formation would increase as increasing IPTG, increasing induction temperature or deceeasing ph value.

中文摘要…………………………………………………… Ⅱ
英文摘要…………………………………………………… Ⅲ
致謝………………………………………………………… Ⅳ
目錄………………………………………………………… Ⅴ
表目錄……………………………………………………… Ⅷ
圖目錄……………………………………………………… Ⅸ
符號表……………………………………………………… Ⅹ


第一章 序論........................................... 1
1-1 前言.............................................. 1
1-2?色螢光蛋白....................................... 2
1-3 細菌外部構造簡介.................................. 6
1-3-1 細菌外部構造................................ 6
1-3-2 革?氏陽性菌和革?氏陰性菌的比較............ 9
1-3-3 革?氏陰性菌外部構造........................ 11
1-4 原生質體的簡介.................................... 17
1-5 革?氏陰性菌原生質體形成方法...................... 19
1-6重組基因的誘導..................................... 21
1-7內涵體(inclusion body)介紹......................... 23
1-8報導基因(reporter gene) ........................... 24
1-9生物螢光影像....................................... 25
1-10研究動機與目的.................................... 26
第二章 實驗材?與步驟................................ 28
2-1實驗材?........................................... 28
2-1-1 藥品........................................ 28
2-1-2 實驗儀器.................................... 30
2-1-3 菌株........................................ 31
2-1-4培養基、緩衝液與Stock solution............... 32
2-2 實驗步驟.......................................... 34
2-2-1 菌株保存與活化............................ 34
2-2-2 原生質體之製備與培養....................... 35
2-2-3 不同培養條件下的內涵體製備................. 36
2-2-4 內涵體的純化............................... 37
2-3 分析方法.......................................... 39
2-3-1原生質體圖像分析............................ 39
2-3-2 內涵體活性分析............................. 40
2-3-3 內涵體比例分析............................ 40
第三章 結果與討論.................................... 41
3-1 內涵體是否有活性.................................. 41
3-2 內涵體的判定及比例分析............................ 44
3-2-1 內涵體的判定............................... 44
3-2-2 內涵體的比例分析........................... 45
3-3 不同培養條件下之內涵體比例........................ 47
3-3-1 誘導溫度對內涵體生成的影響................. 47
3-3-2 誘導劑最終濃度對內涵體生成的影響........... 49
3-3-3 培養液pH值對內涵體生成的影響............... 51
第四章 結論........................................ 53
?考文獻............................................. 55

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