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研究生:楊喬羽
研究生(外文):YANG, CHIAO-YU
論文名稱:增進纖維酒精生產之綜合策略
論文名稱(外文):A combined approach to improve the production of cellulosic ethanol
指導教授:趙雲鵬
指導教授(外文):Chao, Yun-Peng
口試委員:黃光策侯劭毅
口試委員(外文):HUANG, KUANG-TSEHOU, SHAO-YI
口試日期:2017-07-20
學位類別:碩士
校院名稱:逢甲大學
系所名稱:綠色能源科技碩士學位學程
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:99
中文關鍵詞:大腸桿菌纖維酒精引導演化基因重組蛋白代謝工程纖維素酶嗜菌體
外文關鍵詞:Escherichia colicellulosicethanoldirected evolutionGene recombinant proteinmetabolism engineeringCellulasephage
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第一章 緒論
1.1 前言………………................................................……………….......1
1.2 研究動機……………………….......................................................1
第二章 文獻回顧
2.1 生產乙醇之菌株……………………………........................................…......3
2.2 木質纖維素水解液………………………………..........................................….4
2.3 HMF與Furfural抑制機制…………………………….......................................6
2.4 微生物固定化法………………......…………….........................…...............7
2.4.1 包埋法(Entrapment method)………………………..................................…8
2.4.2 海藻酸鈉(Sodium Alginate)…………………..…..................................…8
2.5 纖維素分解……………………………………...............................................…8
第三章 實驗方法
3.1 菌種之保存與活化……………………...……........................................……12
3.1.1 菌種之儲存…………..…………………….............................................12
3.1.2 菌種過夜菌液培養(overnight suspension culture) ….....................12
3.1.3 培養基的配製………………………………............................................…12
3.2 聚合酶連鎖反應 ( Polymerase Chain Reaction, PCR).......................13
3.3 DNA核酸純化方法
3.3.1 質體純化(Purification of plasmid DNA)………............................…14
3.3.2 染色體純化(Purification of chromosome)……………..........................15
3.3.3 瓊脂凝膠萃取DNA片段(DNA extraction from agarose gels)................16
3.3.4 PCR產物純化(Isolation from PCR reactions)……..........…...............17
3.3.5 測定DNA濃度…………………………………..............….....…........................17
3.4 剪切反應 ( Digestion )、連接反應 ( Ligation )、凝膠電泳法 ( Gel Electrophoresis )、In-Fusion…………........……………..........................…..18
3.4.1 剪切反應 ( Digestion )………………………..…...................................18
3.4.2 瓊脂凝膠電泳法 (agarose gel electrophoresis )…….........…............18
3.4.3 連接反應 ( Ligation )…………………..…….................……..................19
3.4.4 In-Fusion……………….………………………..................….......................20
3.5 製備勝任細胞(Competent cell)………………………................................……21
3.5.1 化學法……………………..……………………..........................................….21
3.5.2 電擊穿孔法(Electroporation)…………………................................…….21
3.6 轉形作用( Transformation )…………………..……..............................…...22
3.6.1 化學法製備勝任細胞的轉形…………………………....................................22
3.7 λ Red同源重組(λ Red homologous recombination)…….......…...............23
3.8 位點特異性重組 (Site-specific recombination, SSR)….....................23
3.8.1 FLP-FRT recombination………………...............................…........24
3.8.2 Cre-LoxP recombination……….………......................….............…24
3.8.3 染色體鑲嵌(Integration)…………………..…....................................25
3.9 質體重組與研究之菌株基因型………………….......................................27
3.9.1 pTr-Coh3Ch ........................................................…28
3.9.2 pND-pet…………...………...................................................28
3.9.3 pND-CohII,CBM3,CohI………...…………………...................................…29
3.9.4 pND-ChBD,CohII,CBM3,CohI……………….....................................…30
3.9.5 pND-Coh3Ch…………………………….............................................….31
3.9.6 pPhi-Ter……………………………………..............................................32
3.9.7 pPhi80-WTrHDTCh………………………….................…….....................…..32
3.9.8 pPhi-Coh3Ch……………………………………..................…….......................33
3.9.9 pPhiT7-Coh3Ch…………………………………........................................….34
3.9.10 p15A-mutD5…………………………………............................................35
3.9.11 pSC101-mutD5……………………………….........................................….36
3.9.12 pPR-FabB………………………………….........................……...................37
3.9.13 pTrc-FabA…………………………………............................................…37
3.9.14 pND-LdhA-P80…………………………............................................…38
3.9.15 pAC-GlsD…………………………..............................................….…38
3.9.16 pETD-CelkAD2………………………..............................……..............39
3.9.17 pCelkAD6…………………………….............................................…….40
3.10 纖維素水解液的突變與篩選 ..............................................40
3.11 目標重組蛋白生產檢測...................................................41
3.11.1 蛋白質定量(total protein)...........................................41
3.11.2 蛋白質電泳(SDS-Page)................................................41
3.12 海藻酸鈣複合膠粒固定化.................................................45
3.12.1 海藻酸鈣複合膠粒製備.................................................45
3.12.2 菌種之包埋..........................................................45
第四章 結果與討論
4.1 以六碳糖稻稈纖維素水解液馴化重組菌株…………................................…46
4.2 誘導突變(Mutagenesis)………………..…......................................….…47
4.3 強化脂肪酸代謝或改變脂肪酸結構………….....................................….50
4.4 以突變型DnaQ (mutD5)持續突變及篩選…….....................…..............52
4.5 重組菌株在半纖維稻稈水解液的發酵…………....................…................56
4.6 以六碳糖木片水解液馴化重組菌株……………......................................58
4.7 探討氯化銨做氮源對菌種之影響…………........................................66
4.8 以海藻酸鈣複合膠粒包埋重組菌株並連續批次發酵.…............................69
4.9 胞內生產迷你纖維體…………………………............................................71
4.10 結論與未來展望……………………………...........................................….76
參考文獻……………………………………….................................................………77
附錄......................................................................83
數據分析...................................................................83
標準曲線...................................................................84
實驗材料與器材........................................................................85

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