跳到主要內容

臺灣博碩士論文加值系統

(54.173.214.227) 您好!臺灣時間:2022/01/29 15:47
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:吳孟純
研究生(外文):Meng-Chuen Wu
論文名稱:大腸桿菌巨大原生質體製作及應用之研究
論文名稱(外文):The investingation of generation of giant protoplasts of Escherichia coli and the applied of giant protoplasts
指導教授:鄭智元鄭智元引用關係
指導教授(外文):Chu-Yuan Cheng
學位類別:碩士
校院名稱:國立成功大學
系所名稱:化學工程學系碩博士班
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:2002
畢業學年度:90
語文別:中文
論文頁數:54
中文關鍵詞:原生質體大腸桿菌
外文關鍵詞:protoplastsEscherichia coli
相關次數:
  • 被引用被引用:6
  • 點閱點閱:273
  • 評分評分:
  • 下載下載:45
  • 收藏至我的研究室書目清單書目收藏:0
本研究是利用溶菌酵素(lysozyme)和螯合劑(ethylenediaminetetraacetate),將大腸桿菌JM109宿主細胞製備成原生質體(protoplasts),再將原生質體菌體培養至分別以蔗糖與氯化鉀調配而成的不同滲透壓溶液中,來觀察滲透壓對大腸桿菌原生質體巨大化之影響。吾人發現在以大腸桿菌製備成原生質體時,於SP buffer中添加1% , w/v的EDTA對原生質體的巨大化並無顯著的影響;但卻會造成較高程度之溶菌現象,使得培養液中巨大原生質體菌體數量減少,另外,在培養原生質體時,於GP培養液中添加DNase I (final concen. 17.5 U/mL)對於原生質體的巨大化並無影響,但卻有較高的菌體濃度。在以蔗糖與氯化鉀分別調配不同滲透壓溶液(400 , 700 , 1000 mOsm/Kg H2O)來培養原生質體時發現,滲透壓在原生質體巨大化過程中並沒有明顯變化且其值維持在700 mOsm/kg H2O時,原生質體的數量較多;且粒徑較大,所以可知原生質體巨大化時有一最適滲透壓值,而在相同時間內(16hr),巨大原生質體在同時含有蔗糖與氯化鉀的GP培養液中長得較大且較穩定,菌體數量較能維持。
The present research used Lysozyme and EDTA to produce the protoplasts of Escherichia coli JM109 and incubated protoplasts in medium to form giant protoplasts. The addition of EDTA (1%, w/v) in SP buffer could make high degree of cell lysis and low concentration of protoplasts. On the other hand, the addition of DNase I (final concen. 17.5U/mL) in GP medium could make higher concentration of protoplasts. When protoplasts were incubated in different osmolality medium (400, 700, 1000 mOsm/Kg H2O) with sucrose and KCl to form giant protoplasts, we found that the osmotic pressure of medium wasn’t obvious different during the process and 700 mOsm/Kg H2O of osmolality medium was the optimal condition for protoplasts growing to giant protoplasts. We also found protoplasts could grow greater in size and more stable in GP medium which contend sucrose and KCl during 16 hours.
目錄

中文摘要Ⅰ
英文摘要Ⅱ
目錄Ⅲ
表目錄Ⅴ
圖目錄Ⅵ

第一章 緒論01
1-1前言01
1-2細菌細胞外部構造簡介01
1-2-1細菌細胞外部構造01
1-2-2革蘭氏陰性菌外部構造02
1-3原生質體簡介08
1-4革蘭氏陰性菌形成原生質體的方法08
1-5研究動機與目的14
第二章 實驗材料與方法16
2-1實驗材料16
2-1-1菌株16
2-1-2藥品16
2-1-3培養基17
2-1-4實驗儀器18
2-2實驗方法20
2-2-1菌種保存與活化20
2-2-2宿主細胞之培養20
2-2-3原生質體之製備20
2-2-4原生質體之培養21
2-3分析方法21
2-3-1原生質體濃度測定21
2-3-2原生質體滲透壓測定22
第三章 結果與討論24
3-1確立分析原生質體大小與數量之方法24
3-2 EDTA的添加對巨大原生質體形成之影響29
3-3 DNase I的添加對巨大原生質體形成之影響29
3-4滲透壓對巨大原生質體形成之影響32
第四章 結論49
第五章 未來展望50
參考文獻51
表目錄
表2-1 LB 培養基組成17
表2-2 SP buffer組成17
表2-3 GP 培養基組成18
表3-1 原生質體大小及數量與時間關係表24
表3-2 不同滲透壓之培養液組成35
圖目錄
圖1-1 革蘭氏陰性菌與革蘭氏陽性菌細胞壁構造之區別03
圖1-2 革蘭氏陰性菌細胞外部構造04
圖1-3 Peptidoglycan組成單元06
圖1-4 Peptidoglycan的cross-linkong結構07
圖1-5 LPS layer瓦頂式結構10
圖1-6 親油性物質通過經EDTA作用之外膜之路徑圖12
圖1-7 原生質體形成步驟13
圖3-1 E.coli JM109於GP培養基之生長形態26
圖3-2 OD%、W%與巨大原生質體生長時間關係圖27
圖3-3 OD%、Size%與巨大原生質體生長時間關係圖28
圖3-4 EDTA對E.coli JM109原生質體於GP培養基生長形態之影響30
圖3-5 EDTA對E.coli JM109原生質體於GP培養基生長之影響31
圖3-6 DNase I對E.coli JM109原生質體於GP培養基生長形態之影響33
圖3-7 DNase I對E.coli JM109原生質體於GP培養基生長之影響34
圖3-8 原生質體培養液之滲透壓在菌體巨大化過程中之變化36
圖3-9 以蔗糖改變培養液滲透壓值對E.coli JM109原生質體於培養液中生長形態之影響37
圖3-10以蔗糖改變培養液滲透壓值對E.coli JM109原生質體於培養液中生長之影響39
圖3-11以氯化鉀改變培養液滲透壓值對E.coli JM109原生質體於培養液中生長形態之影響40
圖3-12以氯化鉀改變培養液滲透壓值對E.coli JM109原生質體於培養液中生長之影響41
圖3-13 400 mOsm/Kg H2O滲透壓值對E.coli JM109原生質體於S400,K400培養液中生長形態之影響43
圖3-14 1000 mOsm/Kg H2O滲透壓值對E.coli JM109原生質體於S1000,K1000培養液中生長形態之影響44
圖3-15 700 mOsm/Kg H2O滲透壓值對E.coli JM109原生質體於S700,GP700,K700培養液中生長形態之影響45
圖3-16 400 mOsm/Kg H2O滲透壓值對E.coli JM109原生質體於S400,K400培養液中生長之影響46
圖3-17 1000 mOsm/Kg H2O滲透壓值對E.coli JM109原生質體於S1000,K1000培養液中生長之影響47
圖3-18 700 mOsm/Kg H2O滲透壓值對E.coli JM109原生質體於S700,GP700,K700培養液中生長之影響48
1.Prescott, Harley, Klein, Microbiology, 4th edition, McGraw Hill, New York, p55, 1999.
2.Prescott, Harley, Klein, Microbiology, 4th edition, McGraw Hill, New York, p52, 1999.
3.Bayer.M.E and Leive.L, Effect of ethylenediamineteraacetate upon the surface of Escherichia coli, J. Bacteriol, 130, p1364 ~ 1381, 1977.
4.Birdsell.D.C and Cota-Robles.E.H, Production and Ultrastructure of Lysozyme and Ethylenediaminetetraacetate-Lysozyme Spheroplasts of Escherichia coli, J. Bacteriol, 93, p427 ~ 437, 1967.
5.Iwao Kusaka, Growth and Division of Protoplasts of Bacillus megateriumand Inhibition of Division by Pencillin, J. Bacteriol, 94, p884 ~ 887, 1967.
6.Teuuo Kuroda, Naoyuki Okuda, Naoto Saitoh, Tetsuo Hiyama, etc, Patch Clamp Studies on Ion Pumps of the Cytoplasmic Membrane of Escherichia coli, J. Biological Chemistry, 273, p16897 ~ 16904, 1998.
7.Loek van Alphen, Arie Verkleij, Jose Leunissen-Bijvelt, and Ben Lugtenverg, Architecture of the Outer Membrane of Escherichia coli III. Protein-Lipopolysaccharide Complexes in Intramembraneous Particles, J. Bacteriology, 134, p1089 ~ 1098, 1977.
8.Martti Vaara, Agents That Increase the Permeability of the Outer Membrane, Microbiological Reviews, 56, p395 ~ 411, 1992.
9.Loretta Leive and Virginia K. Shovlin, Physical, Chemical, and Immunological Properties of Lippopolysaccharide Released from Escherichia coli by Ethylenediaminetetraacetate, J. Biological Chemistry, 243, p6384 ~ 6391, 1968.
10.Loretta Leive, Studies on the Permeability Chang Produced in Coliform Bacteria by Ethylenediaminetetraacetate, J. Biological Chemistry, 243, p2373 ~ 2380, 1968.
11.Poirier.I, Marechal.P-A, and Gervais.P, Effects of the kinetics of water potential variation on bacteria viability, Journal of Applied Microbiology, 82, p101 ~106, 1997.
12.Poirier.I, Marechal.P-A, Evrard.C and Gervais.P, Escherichia coli and Lactobacillus plantarum responses to osmotic stress, Appl Microbiol Biotechnol. , 50, p704 ~ 709, 1998.
13.Berner.J.L and Gervais.P, A New Visualization Chamber to Study the Transient Volumetric Response of Yeast Cells Submitted to Osmotic Shifts, Biotechnology and Bioengineering, 43, p165 ~ 170, 1994.
14.Kato.C, Kudo.T, Watanabe.K and Horikoshi.K, Extracellular production of Bacillus pencillinase by Escherichia coli carrying Peap2, Appl Microbio Biotechnol. , 18, p339 ~343, 1983.
15.Ryan.W and Parulekar.S.J, Recombinant protein excretion in Escherichia coli JM103 [pUC8]: effects of plasmid content, ethylenediaminetetraacetate, and phenyl alcohol on cell membrane permeability, Biothechnololgy and Bioengineering, 37, p430 ~ 444, 1991.
16.Boris Martinac, Matthew Buechner, etc, Pressure-sensitive ion channel in Escherichia coli, Proc.Natl.Acad.Sci.USA, 84, P2297 ~ 2031, 1987.
17.Andrzej Kubalski, Generation of giant protoplasts of Eschericha coli and an inner-membrane anion selective conductance, Biochimica et Biophysica Acta, 1238, p177 ~ 182, 1995.
18.Wei Hu and Chi-Lien Cheng, Expression of Aequorea green fluorescent protein in plant cells, FEBS Letters, 369, p331 ~334, 1995.
19.Pilar Fernandez de Palencia, Concha Nieto, Paloma Acebo, Manuel Espinosa and Paloma Lopez, Expression of green fluorescent protein in Lactococcus lactis, FEMS Microbiology Letters, 183, p229 ~ 234, 2000.
20.Jonathan Porter, Roger Pickup and Clive Edwards, Flow cytometric detection of specific genes in genetically modified bacteria using in situ polymerase chain reaction, FEMS Microbiology Letters, 134, p51 ~ 56, 1995.
21.Jakob Moller-Jensen, Jensen R.B and Kenn Gerdes, Plasmid and chromosome segregation in prokaryotes, Trends in Microbiology, 8, p313 ~320, 2000.
22.Frederick C. Neichardt, Escherichia coli and Salmonella typhimurium: cellular and molecular biology, V1, American society for microbiology, N.W, 1987.
23.Frederick C. Neichardt, John L. Ingraham and Moselio Schaechter, Physiology of the Bacterial Cell: A Molecular Approach, Sinauer Associates Inc, Sunderland, Massachusetts, U S A, 1990.
24.Mary A. Asbell and Eagon R.G, Role of Multivalent Cations in the Organization, Structure, and Assembly of the Cell Wall of Pseudomonas aeruginosa, Journal of Bacteriology, 92, p380 ~ 387, 1966.
25.Sambrook J., Fritsch E.F and Maniatis T., Molecular Cloning, 2nd edition, Cold Spring Harbor Laboratory Press, U S A, 1989.
26.Wolfgang Epstein and Stanley G. Schultz, Cation Transport in Escherichia coli V. Regulation of cation content, The Journal of General Physiology, 49, p221 ~ 234, 1965.
27.Ferris F.G. and Beveridge T.J., Physicochemical roles of soluble metal cations in the outer membrane of Escherichia coli K-12, CAN. J. Microbiol. , 32, p594 ~ 601, 1986.
28.Binding, H. and Reinert, J., Differentiation of protoplasts and of transformed plant cells, Springer-Verlag, N.W., 1983.
連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top