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研究生:馬經綸
研究生(外文):Jing-lun Ma
論文名稱:酵母菌之木質酒精生產的先期研究
論文名稱(外文):Preliminary study of xylulosic ethanol production by yeast
指導教授:趙雲鵬
指導教授(外文):Yun-Peng Chao
學位類別:碩士
校院名稱:逢甲大學
系所名稱:化學工程學所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:105
中文關鍵詞:酒精麵包酵母菌代謝工程基因工程木醣
外文關鍵詞:Saccharomyces cerevisiaeGenetic engineeringMetabolic engineeringEthanolXylose
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中文摘要
為了發展麵包酵母菌之木糖醱酵生成酒精的技術平台,在本研究中,我們採用之主要策略包括(1)突變Pichia stipitis XR為 NADH消耗倚賴性 和 突變Pichia stipitis XDH為 NADPH生成倚賴性, (2)發展染色體基因插入方法(3)建構剔除染色體上抗抗生素基因的質體。實驗結果顯示,突變型P. stipitis XR、XDH確實可以改變其對於氧化還原分子的依賴性,唯酵素活性也因之而降低。此外,運用發展之染色體基因插入法,我們成功的將突變型Pichia stipitis XR和XDH鑲箝入麵包酵母菌染色體上,如此將可以達到穩定轉殖表現Pichia stipitis XR、XDH之目的,並且嘗試剔除染色體上的抗抗生素基因,來幫助實驗上的發展。
Abstract

The main purpose of this work is to develop a technology platform for conversion of xylose to ethanol in baker yeast. To achieve this goal, three main strategies were adopted including (1) creation of NADH-dependent Pichia stipitis XR by mutation, and generation of NADP-dependent Pichia stipitis XDH by mutation, (2) development of the genomic gene insertion method, (3) construct plasmid that can be use in delet marker when that in chromosome. As a result, Pichia stipitis XR and XDH were mutated to shift their respective redox preference while their specific activities were accordingly reduced. Moreover, the mutant form of Pichia stipitis XR was successfully inserted into yeast genome by the developed method. This will permit a stable expression of inserted genes in the cell and also try to delet the DNA marker that from the yeast genome.
目 錄
第一章 緒論
1.1 前言……………………………………………………………… 1.
1.2 文獻回顧………………………………………………………… 2.
1.3 研究動機………………………………………………………… 5
第二章 實驗方法
2.1 菌種之儲存與馴養…………………………………………….. 7.
2.1.1 菌種儲存……………………………………………..……. 7.
2.1.2 菌種馴養…………………………………………………... 7.
2.1.3 培養機的配製……………………………………………. 7.
2.2 DNA 純化方法……………………………………………….. 8.
2.2.1 質體純化……………………………….………………… 8.
2.2.2 瓊脂凝膠萃取DNA 片段………………………………. 10.
2.2.3 PCR DNA 純化……………………….…………………. 11.
2.2.4 DNA濃度測量…………………..……………………….. 12.
2.3 染色體純化方法……………………………………………… 13
2.4 剪切反應、連接反應、凝膠電泳法………………………… 15.
2.4.1 剪切反應…………………………………………………. 15.
2.4.2 凝膠電泳法………………………………………………. 16.
2.4.3 連接反應…………………………………………………. 17.
2.5 酒精沈澱……………………………………………………… 18.
2.6 大腸桿菌勝任細胞的準備…………………………………… 18.
2.6.1 化學法……………………………………………………. 18.
2.6.2 電擊穿透法………………………………………………. 19.
2.7 麵包酵母菌勝任細胞的準備………………………………… 20.
2.7.1 化學法……………………………………………………… 20
2.7.2 電擊穿透法………………………………………………… 21
2.8 轉殖作用(Transformation) ………………………………………22
2.8.1 大腸桿菌以化學法製備勝任細胞的轉殖………………. 22.
2.8.2 大腸桿菌以電擊穿透法製備勝任細胞的轉殖…………. 22.
2.8.3 酵母菌株以化學法製備勝任細胞的轉殖………………… 24
2.8.4 酵母菌株以電擊穿透法製備勝任細胞的轉殖…………… 24
2.9 系統檢測………………………………………………… …... 25.
2.9.1 蛋白質定量……………………………………………… 25.
2.9.2 蛋白質電泳(SDS-PAGE)……………………….. ….. …..25.
2.10 聚合酵素連鎖反應(Polymerase Chain Reaction)………… 28
2.10.1 大量複製所欲之特定基因(PCR Amplification)………29
2.10.2 定點突變(QuikChangeR Site-Directed mutagenesis kit)…30
2.11 重組菌種製備…………………………………………………32
2.12 HPLC及GC分析……………………………………………… 32
2.12.1 分析碳源……………………………………………………32
2.12.2 分析酒精……………………………………………………33
2.13 重組質體之建構……………………………………………… 34
2.13.1 建構基因表現質體…………………………………………34
I. 建構質體pXYL1和pXYL1M…………………………………34
II. 建構質體pXYL2和pXYL2M4………………………………35
2.13.2 基因表現質體活性測試……………………………………35
I. 酵素活性測試方法(Acativity assay) …………………………35
II. 檢測P. stipitis XYL2、XYL2M4基因的活性………………37
III. 檢測P. stipitis XYL1、XYL1M基因的活性…………………37
2.14 建構基因鑲箝染色體工具之質體 …………………………… 38
2.14.1 質體pGK-XYL1M…………………………………………38
2.14.2 質體pHO-PGK/XYL2b與pHO-pGK/XYL2M4b…………38
2.15 外源基因鑲箝至染色體(Genomic insertion)…………………39
2.15.1 P. stipitis XYL1M基因鑲箝至麵包酵母菌染色體 ………39
2.15.2 P. stipitis XYL2M4基因鑲箝至麵包酵母菌染色體………40
2.15.3 檢測P. stipitis XYL2、XYL2M4基因鑲嵌至酵母菌株染色
體的活性……………………………………………………41
2.16 外源基因XDH與XR同時作用於酵母菌株之測試…………42
2.16.1 Candida guilliermondii 質體pCXYL1……………………42
2.16.2 檢測P. stipitis XR、Candida guilliermondii CXYL1基因的
活性…………………………………………………………42
2.17 建構剔除Marker之質體………………………………………43
2.17.1 質體pMCRE-B……………………………………………43
2.17.2 質體pMCRE-K……………………………………………43
2.18 剔除重組基因菌株染色體上的抗抗生素基因……………… 44
第三章 結果與討論
3.1 基因表現質體活性測試與液態基質培養測試………………45
3.1.1 檢測外源P. stipitis XR與XDH基因的活性. …………45
3.1.2 檢測P. stipitis XYL1、XYL1M基因的活性……………46
3.2 外源基因鑲嵌至染色體後的測試. ………………………... 47.
3.2.1 檢測P. stipitis XYL2、XYL2M4基因鑲嵌至酵母菌株染色體
的活性……………………………………………………. 47
3.2.2 檢測以P. stipitis XYL2M4基因鑲嵌至酵母菌株染色體為載
體來比較P. stipitis XYL1 與Candida guilliermondii CXYL1
基因的生長情況與活性……………………………………48
3.2.3 檢測以Pichia stipitis XYL2基因鑲嵌至酵母菌株染色體為載
體來比較P. stipitis XYL1 與C. guilliermondii CXYL1基因的生
長情況與活性…………………………………………………52
第四章 結論與未來展望…………………………………………... 53.
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