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研究生： 吳孟純
Wu, Meng-Chuen
論文名稱： 大腸桿菌巨大原生質體製作及應用之研究
The investingation of generation of giant protoplasts of Escherichia coli and the applied of giant protoplasts
指導教授： 鄭智元
Cheng, Chu-Yuan
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： 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： protoplasts, Escherichia coli
相關次數： 點閱：71  下載：2 
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    • 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

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