本研究以Escherichia coli JM109/pSCR05探討維持質體穩定性的醱酵策略。有鑑於質體脫落的時機在細胞分裂時，因此吾人提出降低細胞分裂機率的兩階段醱酵策略以維持質體穩定性。其策略為第一段以適合大腸桿菌生長的培養基(生長培養基)提高菌體濃度，在第二階段加入適合肌酸酵素生產之培養基(生產培養基)與誘導劑isopropyl-β-D–thiogalactopyranoside (IPTG) 誘導肌酸酵素生產。由於菌體量倍增的時機意謂醱酵槽內大部分細胞已經過一次分裂產生新生代。所以，藉由調整生產培養基的添加量，可控制誘導後菌體的增加量，減少細胞分裂機率，達到維持質體穩定性的目標。

　　A fermentation strategy for maintaining plasmid stability was investigated in this study. Since plasmid loss occurs during cell fission, a two-stage fermentation method for decreasing the probability of cell fission was proposed accordingly. At the first stage, high cell density was achieved by culturing the cells in the growth medium. At the second stage, the production of creatinase was produced by feeding the fresh producing medium with a appropriate concentration of isopropyl-β-D–thiogalactopyranoside (IPTG). The timing of cell mass doubled implied that the majority of cells had experienced one cycle of cell fission and new generation of cells were born. Therefore, the maintenance of plasmid stability could be achieved by decreasing the occurrence of cell fission, which can be done by adjusting the adding of producing medium for controlling the increase of cell mass after induction.

目錄

中文摘要………………...………………………………………………..…Ⅰ
英文摘要………………...………………………………………………..…Ⅱ
誌謝…………………………...…………………………………………..…Ⅲ
目錄…………………………...…………………………………………..…Ⅴ
表目錄…………………………...…………………………………..………Ⅶ
圖目錄……………………………...…………………………………..……Ⅷ

第一章 緒論……………...…………………………………………………1
1-1前言………………………………………...……………………………..1
1-2誘導機制…………………………………...…………………….……….2
1-3 肌酸酵素…………………………………………………..……….…….4
1-4 質體複製數和質體穩定性…………......……………………………6
1-5 醱酵策略…………………………………………………………….7
1-6 研究目的…………………………….………………………………….10

第二章 實驗材料和方法……………...…….…………………………….11
2-1菌株及藥品……………………………………………………………...11
2-1-1 宿主-載體系統……………………...………………………..11
2-1-2 藥品………………………………...………………………...13
2-2儀器與裝置……………………………………………..………………14
2-3實驗方法………………………………..………………………………16
2-3-1 菌種的保存…………………………………………………...16
2-3-2 搖瓶振盪培養………………………………………………...16
2-3-3 醱酵槽實驗…………………………………………………...18
2-4分析方法……………………………………………………..…………22
2-4-1 菌體濃度分析方法………….………………………………...22
2-4-2 肌酸酵素活性分析..……….……………….………………..25
2-4-3 質體穩定性分析..…………….…..………….………………27

第三章 結果與討論…………………………..….…………………………29
3-1培養基的選擇………………………….……….…………….………....29
3-2二階段批次醱酵…………………………...……………………………34
3-3高質體穩定性之原因探討……………………..……………………….40
3-4饋料批次(fed-batch)醱酵…………………………….…………………45

第四章 結論……………………..……………….…………………………50
參考文獻………………………………..…………….……………………..51
自述……………………………………..…………….……………………..53

參考文獻

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王憲忠，菌體外分泌蛋白質－幾丁分解酶之基因解析，國立成功大學生物化學研究所碩士論文(1993)。