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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳俊任 | |
| dc.contributor.author | Chu-Chiao Chin | en |
| dc.contributor.author | 秦楚喬 | zh_TW |
| dc.date.accessioned | 2021-06-13T06:34:08Z | - |
| dc.date.available | 2016-10-21 | |
| dc.date.copyright | 2011-10-21 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-09-05 | |
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Brestic-Goachet N, Gunasekaran P (1989) Transfer and Expression of an Erwinia chrysanthemi Cellulase Gene in Zymomonas mobilis. Journal of General Microbiology 135: 893-902. 46. Yoon KH, Park lH, Pack MY (1988) Transfer of Bacillus Subtilis Endo-β-1,4-Glucanase Gene Into Zymomonas Anaerobia. Biotechnology Letters 10: 213-216. 47. Lejeune A, Eveleigh DE, Colson C (1988) Expression of an endoglucanase gene of Pseudomonas fluorescens var. cellulosa in Zymomonas mobilis. FEMS Microbiology Letters 49: 363-366. 48. Su P, Delaney SF, Rogers PL (1989) Cloning and expression of a β-glucosidase gene from Xanthomonas albilineans in Escherichia coli and Zymomonas mobilis Journal of Biotechnology 9: 139-152. 49. Yanase H, Masuda M, Tamaki T (1990) Expression of the Escherichia coli α-Galactosidase and Lactose Permease Genes in Zymomonas mobilis and Its Raffinose Fermentation Journal of Fermentation Bioengineering 70: 1-6. 50. Yanase H, Kotani T, Yasuda M, Matsuzawa A (1991) Metabolism of galactose in Zymomonas mobilis. Appl Microbiol Biotechnol 35: 364-368. 51. Buchholz SE, Dooley MM, Eveleigh DE (1989) Growth of Zymomonas on lactose: gene cloning in combination with mutagenesis Journal of Industrial Microbiology 4: 19-27. 52. Lodgea J, Feara J, Busby S, Gunasekaranb P (1992) Broad host range plasmids carrying the Escherichia coli lactose and galactose operons. FEMS Microbiology Letters 95: 271-276. 53. Liu C-Q, Goodman AE, Dunn NW (1988) Expression of cloned Xanthomonas D-xylose catabolic genes in Zymomonas mobilis Journal of Biotechnology 7: 61-70. 54. Zhang M, Eddy C, Deanda K, Finkelstein M, Picataggio S (1995) Metabolic Engineering of a Pentose Metabolism Pathway in Ethanologenic Zymomonas mobilis. Science 267: 240-242. 55. Mackenzie KF (1989) Modulation of alcohol dehydrogenase isoenzyme levels in Zymomonas mobilis by iron and zinc. JOURNAL OF BACTERIOLOGY 171: 1063-1067. 56. Kono M (1990) Purification and some properties of chitinase from the stomach of Japanese Eel, Anguilla japonica. Agric Biol Chem 54: 973-978. 57. Roberts WK (1988) Plant and bacterial chitinases differ in antifungal activity. Journal of General Microbiology 134: 169-176. 58. Koga D (1989) Kinetics of chitinase from yam, Dioscorea opposita THUNB. Agric Biol Chem 53: 3121-3126. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34739 | - |
| dc.description.abstract | Zymomonas mobilis 為一革蘭氏陰性菌，具有良好的酒精生產能力而在生質酒精發展中受到重視。Z. mobilis 和一般常用來生產酒精之酵母菌相比，有較高的酒精產量和較高的酒精耐受性等優勢，然而 Z. mobilis 可代謝之基質範圍狹窄，僅能使用葡萄糖、蔗糖和果糖，因此在應用上受到限制。幾丁質是由 N -乙醯葡萄糖胺 (GlcNAc) 聚合而成的分子，其含量僅次於纖維素，是自然界中含量第二高的多醣類，故具有作為生質酒精基質的潛力。 Z. mobilis 中缺乏代謝 GlcNAc 所需之基因，故無法順利使用 GlcNAc 作為其生長所需之碳源。我們試圖使 Z. mobilis 得以利用 GlcNAc 作為其生長之碳源，因此在 Z. mobilis 中建構並表現 Escherichia coli 的三個基因 (nagA, nagB, nagE)，此三個基因分別可轉譯出 GlcNAc6P deacetylase 、 GlcN6P deaminase 和 GlcNAc 的運輸蛋白。在基因的建構設計上，我們參考乳糖操作組的模式，在相連接的每段基因中額外設計了一組 RBS 序列。本研究中，我們複製了這三個基因並且成功的送入 pET-28a 載體中。起初在基因的表現上遭遇了一些問題，但在經過多次調整後，我們成功在 E. coli 中表現了這三個蛋白。現在我們嘗試將這三個基因次轉殖送入可在 Z. mobilis 中表現異源蛋白的載體 pKT230 。最後，我們期望所建構的 Z. mobilis 得以利用 GlcNAc 做為其生長所需之碳源而生產酒精。 | zh_TW |
| dc.description.abstract | Zymomonas mobilis is a gram-negative bacterium which is notable for its bioethanol-producing capabilities. Compared to yeasts, Z. mobilis has better ethanol tolerance and higher ethanol yield. One limitation of Z. mobilis is that it can only use glucose, fructose, and sucrose as substrates for ethanol fermentation. Chitin is a polysaccharide consisting of N-acetylglucosamine (GlcNAc) monomers, and it is the second most abundant polysaccharide after cellulose on earth. Chitin can be hydrolyzed into GlcNAc by microbial chitinases, and the aim of this study is to engineer Z. mobilis to utilize GlcNAc for bioethanol production. Our strategy is to clone and express three E. coli genes:NagE, NagA,and NagB in Z. mobilis. NagE encodes a GlcNAc transporter, NagA encodes a GlcNAc-6-phosphatedeacetylase, and NagB encodes aglucosamine-6-phosphate deaminase. We have successfully cloned these three genes and expressed them in E. coli. By placing a ribosome binding site (RBS) in front of each gene, we have engineered a bicistronic construct that can co-express NagA and NagB. We have also successfully cloned and expressed NagE, which is a membrane protein and can only be detected in the enriched membrane fraction. The cloned NagA, NagB in E. coli, and NagE genes are being subcloned into the broad-host-range plasmid pKT230 and will be expressed in Z. mobilis. The use of GlcNAc for ethanol fermentation in the engineered Z. mobilis will be further investigated. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T06:34:08Z (GMT). No. of bitstreams: 1 ntu-100-R98b47110-1.pdf: 5078113 bytes, checksum: 4cd5f3f0accd403f14e7e181e8baa88a (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | 目錄
中文摘要 ........................................................................................................................... I Abstract ............................................................................................................................. II 縮寫表 ............................................................................................................................ IV 專有名詞 中英文對照表 ................................................................................................ V 目錄 ............................................................................................................................... VII 圖表目錄 .......................................................................................................................... X 第一章 前言 ................................................................................................................... 1 1.1 生質能源 ......................................................................................................... 1 1.1.1 以糖類做為基質醱酵生質酒精 ............................................................. 2 1.1.2 以澱粉做為基質醱酵生質酒精 ............................................................. 2 1.1.3 以纖維素做為基質醱酵生質酒精 ......................................................... 3 1.1.4 生質酒精所面臨之相關問題 ................................................................. 4 1.2 以 Zymomonas mobilis 進行酒精醱酵......................................................... 4 1.3 幾丁質 ............................................................................................................. 5 1.4 nag 基因 ......................................................................................................... 6 1.5 實驗目的與架構 ............................................................................................. 7 第二章 實驗材料與方法 ................................................................................................. 8 2. 1 Z. mobilis 於 GlcNAc 中之生長與酒精濃度測試 ..................................... 8 2.1.1 Z. mobilis 培養 ....................................................................................... 8 2.1.2 酒精濃度之測量 ..................................................................................... 9 2.2 基因之建構 ....................................................................................................... 9 2.2.1 菌株與質體 ............................................................................................... 9 2.2.2 E. coli 基因體 DNA 之萃取 .............................................................. 11 2.2.3 以 PCR 方法製備基因片段 ............................................................... 12 2.2.4 轉形與篩選 ........................................................................................... 13 2.2.5 質體之萃取與限制酶剪切之檢驗 ....................................................... 13 2.2.6 質體之建構 ........................................................................................... 14 2.2.6.1 pET-NagB 質體之建構 ................................................................ 14 2.2.6.2 pET-NagA 質體之建構 ................................................................ 15 2.2.6.3 pET-NagE 質體之建構 ................................................................. 15 2.2.6.4 pET-NagBA 質體之建構 .............................................................. 16 2.2.6.5 pKT-NagBA 質體之建構 ............................................................. 16 2.2.6.6 pKT-NagE 質體之建構 ................................................................. 16 2.2.6.7 pET-NagE (single RBS) 質體之建構 ........................................... 17 2.2.6.8 pET-NagE (ΔHis-T7)質體之建構 ................................................. 17 2.3 蛋白質之表現 ............................................................................................... 17 2.3.1 SDS-PAGE ............................................................................................ 17 2.3.2 Coomassie Brilliant Blue (CBR) 蛋白質染色法 ................................. 18 2.3.3 西方墨點法 ........................................................................................... 19 2.3.4 膜蛋白之萃取 ....................................................................................... 19 第三章 實驗結果 ........................................................................................................... 21 3.1 Zymomonas mobilis 於 GlcNAc 中的生長情形與酒精產量 ................... 21 3.2 pET-NagB、pET-NagA 及 pET-NagE 之基因建構 ................................. 21 3.2.1 pET-NagB 之基因建構 ....................................................................... 21 3.2.2 pET-NagA 之基因建構 ....................................................................... 22 3.2.3 pET-NagE 之基因建構 ........................................................................ 23 3.3 各基因於 E. coli BL21 (DE3) 中之表現 ................................................... 23 3.3.1 pET-NagB 於 E. coli BL21 (DE3) 中之表現 .................................... 23 3.3.2 pET-NagA 於 E. coli BL21 (DE3) 中之表現 .................................... 24 3.3.3 NagB 與 NagA 同時於 E. coli BL21 中之表現 ............................. 24 3.3.4 NagE 於 E. coli BL21 中之表現 ....................................................... 25 第四章 討論 ................................................................................................................... 26 圖表 ................................................................................................................................. 30 文獻參考 ......................................................................................................................... 59 | |
| dc.language.iso | zh-TW | |
| dc.subject | 乳糖操作組 | zh_TW |
| dc.subject | Zymomonas mobilis | zh_TW |
| dc.subject | N -乙醯葡萄糖胺 | zh_TW |
| dc.subject | nagA | zh_TW |
| dc.subject | nagB | zh_TW |
| dc.subject | nagE | zh_TW |
| dc.subject | Zymomonas mobilis | en |
| dc.subject | RBS | en |
| dc.subject | NagB | en |
| dc.subject | NagA | en |
| dc.subject | NagE | en |
| dc.subject | N-acetylglucosamine | en |
| dc.title | 建立 Escherichia coli NAG 基因表達系統之研究 | zh_TW |
| dc.title | Development of the expression system for Escherichia coli NAG genes | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 吳蕙芬,楊啟伸,林乃君 | |
| dc.subject.keyword | Zymomonas mobilis,N -乙醯葡萄糖胺,nagA,nagB,nagE,乳糖操作組, | zh_TW |
| dc.subject.keyword | Zymomonas mobilis,N-acetylglucosamine,NagE,NagA,NagB,RBS, | en |
| dc.relation.page | 63 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2011-09-05 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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