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研究生: 謝曜駿
Xie, Yao Jun
論文名稱: 以生物取像系統探討綠色螢光蛋白在不同培養條件下之基因重組大腸桿菌內涵體的形成
Study of Green Fluorescence Protein Inclusion Body Formation under Various Culture Conditions in Recombinant Escherichia coli with Bioimaging System
指導教授: 鄭智元
Zheng, ZiYuan
學位類別: 碩士
Master
系所名稱: 工學院 - 化學工程學系
Department of Chemical Engineering
論文出版年: 2010
畢業學年度: 98
語文別: 中文
論文頁數: 60
中文關鍵詞: 綠色螢光蛋白大腸桿菌巨大原生質體生物取向系統內涵體
外文關鍵詞: GFP, inclusion body, Escherichia coli
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  • 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綠色螢光蛋白....................................... 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 革蘭氏陽性菌和革蘭氏陰性菌的比較............ 9 1-3-3 革蘭氏陰性菌外部構造........................ 11 1-4 原生質體的簡介.................................... 17 1-5 革蘭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 實驗材料與步驟................................ 28 2-1實驗材料........................................... 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 參考文獻............................................. 55

    1. Baumann, P., L. Baumann, M. J. Woolkalis, and S. S. Bang, “Evolution relationship in Vibrio and Photobacterium: a basis for a natural classification,” Annu. Rev. Microbial., 37: 369-398 (1983).
    2. Birdsell, D. C., and E. H. Cota-Robles, “Production and ultrastructure of lysozyme and ethylenediaminetetraacetate-lysozyme Spheroplasts of Escherichia coli,” J. Bacteriol., 93: 427-437 (1967).
    3. Bullen, J. J., Spalding, P. B., Ward, C. G., and Gutteridge,
    “Hemochromatosis, iron and septicemia caused by Vibrio vulnificus. Arch.Intern.Med.” J. M. 151:1606-1609 (1991).
    4. Campbell, A.K., “Living light: biochemistry, function and biomedical application,” Essays Biochem., 24: 41-81 (1989).
    5. Chang, C. C., Y. C. Chuang, Y. C. Chen, and M. C. Chang, “Bright fluorescent of a novel protein from Vibrio vulnificus depends on NADPH and the expression of this protein is regulated by a LysR-type regulatory gene,” Biochem. Biophys. Res. Commun., 319: 207-213 (2004).
    6. Chang, C. C., Y. C. Yin, and M. C. Chang, “Fluorescent intensity of a novel NADPH-binding protein of Vibrio vulnificus can be improved by directed evolution,” Biochem. Biophys. Res. Commun., 322: 303-309 (2004).
    7. Costerton, J. W., J. M. Ingram, and K. J. Cheng, “Structure and function of cell envelope of Gram-negative bacteria.”
    Bacteriolgical Reviews, 87-110 (1974).
    8. Epstein, W., and S. G. Schultz, “Cation transport in Escherichia coli V.Regulation of cation content”, J. Gen. Physiol., 49: 221-234 (1965).
    9. Furtado, Agnelo, and Robert Henry, “Measurement of green fluorescent protein concentration in single cells by image analysis,” Anal. Biochem., 310:84-92 (2002).
    10. Gabriel D. Peckham, Robert C. Bugos, Wei Wen Su, “PuriWcation of GFP fusion proteins from transgenic plant cell cultures,” Protein Expression and PuriWcation., 49: 183–189 (2006).
    11. Hastings, J. W., C. J. Potrikus, S. C. Gupta, M. Kurfurst, and J. C.Makemson, “Biochemistry and physiology of bioluminescent bacteria,” Adv. microb. physiol., 26: 235-291 (1985).
    12. Heeswijk, W. C., Rabenberg, M., Weterhoff, H. V., and Kahn, D.“The genes of the glutamine systhetase adenylation cascade are not regulated by nitrogen in E. coli. Mol.,” Microbiol., 9:443-457(1993).
    13. Hsu, R. Y., and H. A. Lardy, “Cleland WW. Pigeon liver malic enzyme.V. Kinetic studies,” J. Biol. Chem., 242: 5315-5322 (1967).
    14. Hu, w. and C. C. Cheng, “Expression of aequorea green fluorescent protein in plant cells.” FEBS Letters., 369: 331-334 (1995).
    15. Kahana, J., and P. Silver, “Current protocols in molecular biology,” In Ausabel, F.(ed). Green and Wiley, N.Y. (1996).
    16. ornvall, H., B. Persson, M. Krook, S. Atrian, R. Gonzalez-Duarte, J.Jeffery, and D. Ghosh, “Short-chain dehydrogenases/reductases (SDR),”Biochemistry,34: 6003-6013 (1995).
    17. Kuroda, T., N. Okuda, N. Saitoh, T. Hiyama, Y. Terasaki, H.Anazawa, A. Hirata, T. Mogi, I. Kusaka, T. Tsuchiya, I. Yabe, “Patch clamp studies on ion pumps of the cytoplasmic membrane of Escherichia coli,” J. Biol. Chem., 273: 16897-16904 (1998).
    18. Kusaka, I., “Growth and division of protoplasts of bacillus megateriumand inhibition of division by pencillin,” J. Bacteriol., 94: 884-887(1967).
    19. Leive, L., “Studies on the permeability chang produced in coliform bacteria by ethylenediaminetetraacetate,” J. Biol. Chem., 243: 2373-2380(1968).
    20. Libby G. Puckett, Jennifer C. Lewis, Leonidas G. Bachas,* and Sylvia Daunert,“Development of an assay for ß-lactam hydrolysis using the pH-dependence of enhanced green fluorescent protein,” Analytical Biochemistry., 309: 224–231 (2002).
    21. Li, B., and S. X. Lin, “Fluorescence-energy transfer in human estradiol 17 beta-dehydrogenase-NADPH complex and studies on the coenzyme binding,” Eur. J. Biochem., 235: 180-186 (1996).
    22. Matz, M. V., A. F. Fradkov, Y. A. Labas, A. P. Savitsky, A. G.Zaraisky, M. L. Markelov, and S. A. Lukyanov, “Fluorescent proteins from nonbioluminescent Anthozoa species,” Nat. Biotechnol., 17: 969-973 (1999).
    23. McDougald D., L. M. Simpson, J. D. Oliver, and M. Hudson,
    “Transformation of Vibrio vulnificus by electroporation,” Curren. Microbiol.,28: 289-291 (1994).
    24. Meighen, E. A.,“Molecular biology of bacterial bioluminescence,”Microbiol Rev., 55: 123-142 (1991).
    25. Meighen, E. A., and I. Bartlet, “Complementation of subunits from different bacterial luciferases. Evidence for the role of the beta subunit in the bioluminescent mechanism,” J. Biol. Chem., 255: 11181-11187 (1980).
    26. Miksch, G., F. Bettenworth, K. Friehs, and E. Flaschel, “The sequence upstream of the -10 consensus sequence modulates the strength and induction time of stationary-phase promoters in Escherichia coli.” Appl. Microbiol.Biotechnol., 69: 312-320 (2005).
    27. Miksch, G., F. Bettenworth, K. Friehs, E. Flaschel, A. Saalbach, and T. W. Nattkemper, “A rapid reporter system using GFP as a reporter protein for identification and screening of synthetic stationary-phase promoters in Escherichia coli.” Appl. Microbiol. Biotechnol., 69: 1-8 (2005).
    28. Morin, J. G., and J. W. Hastings, ”Energy transfer in a bioluminescent system,” J. Cell Physiol., 77: 313-318 (1979).
    29. Morise, H., O. Shimomura, F. H. Johnson, and J. Winant,
    “Intermolecular energy transfer in the bioluminescent system ,” Aequorea.Biochemistry., 13: 2656-2662 (1974).
    30. Paparella, M., E. Kolssov, B.K. Fleischmenn, J. Hescheler, and S.Bremer, “The use of quantitative image analysis in the assessmentof in vitro embryotoxicity endopoints based on a novel embryonic stem cell clone with endoderm-related GFP expression,” Toxicol. Vitro, 16: 589-597 (2002).
    31. Prakash, Y. S, “Fluorescent and Luminescent Probs,” 2nd ed., Academic press, 316-330 (1999).
    32. Prescott, Harley, Klein, Microbiology, 5th edition, McGraw Hill, New York, 56-59 (2001).
    33. Ran Zhuang, Yuan Zhang, Rui Zhang, Chaojun Song, Kun Yang,
    Angang Yang, Boquan Jin, “Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography,” Protein Expression and Purification., 59: 138–143 (2008).
    34. Schmidt, T. M. , K. Kopecky, and K. H. Nealson, Appl. Environ.Microbiol., 55: 2607-2612 (1989).
    35. Shelley R. McRae, Christopher L. Brown, Gillian R. Bushell, “Rapid puriWcation of EGFP, EYFP, and ECFP with high yield and purity,” Protein Expression and Purification., 41: 121–127 (2005).
    36. Siegele, D. A., L. Campbell, and J. C. Hu, “Green fluorescent protein as a reporter of transcriptional activity I prokaryotic system,” Methods Enzymol.,305: 499-513 (2000).
    37. Su, J.H., Y. C. Chung, Y. C. Tsai, and M. C. Chang, “Cloning and characterization of a blue fluorescent protein from Vibrio vulnificus,”Biochem. Biophys. Res. Commun., 287: 359-365 (2001).
    38. Tacket, C. O., F. Brenner, and P. A. Blake, “Clinical features and an epidemiological study of Vibrio vulnificus infections,” J. Infect. Dis., 149:558-561 (1984).
    39. Youvan, D.C. and M. E. Michel-Beyerle, “Structure and fluorescence mechanism of GFP,” Nat. Biotechnol., 14: 1219-1220 (1996).
    40. Yang, F., L.G. Moss, and G. N. Jr. Phillips, “The molecular structure of green fluorescent protein.” Nat. Biotechnol., 14: 1246-1251 (1996).
    41.李瑞俞, 以生物取像系統探討在不同培養條件下之基因重組大腸
    桿菌內涵體的形成, 成功大學化學工程研究所碩士論文, 1-21 (2007).
    42.袁道成, 創傷桿菌感測及反應雙因子調控系統對血清、抗菌蛋白及嗜中性球抗性之探討, 成功大學生物化學暨分子生物學研究所碩士論文,22-56 (2005).
    43.陳俊吉, 利用回應曲面法探討添加胺基酸對肌酸酵素生產之影響,成功大學化學工程研究所碩士論文, 3-6 (2003).
    44.劉再鈜, 創傷弧菌藍色螢光蛋白應用於革蘭氏陰性菌巨大原生質體發光特性之研究, 成功大學化學工程研究所碩士論文, 1-18 (2006).108
    45.劉智偉, 創傷弧菌之藍色螢光蛋白應用於革蘭氏陰性菌生物取像系統之研究, 成功大學化學工程研究所碩士論文, 15-27 (2005).
    46.鄧怡芳, 創傷弧菌致病因子之研究, 成功大學生物科技研究所碩士論文, 7-30 (2004).
    47.翁偉程, 綠色螢光蛋白在基因重組大腸桿菌生物取向系統應用之研究, 成功大學化學工程研究所碩士論文, 1-20(2009)

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