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研究生: 陳義洋
Yi-Yang Chen
論文名稱: 利用原子力顯微鏡在溶液中探討線狀噬菌體和大腸桿菌之交互作用
Investigate the Interaction between Filamentous Bacteriophage and Escherichia coli by Atomic Force Microscopy in Liquid
指導教授: 游萃蓉
Tri-Rung Yew
口試委員:
學位類別: 碩士
Master
系所名稱: 工學院 - 材料科學工程學系
Materials Science and Engineering
論文出版年: 2008
畢業學年度: 96
語文別: 中文
論文頁數: 69
中文關鍵詞: 原子力顯微鏡噬菌體大腸桿菌
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  • 本研究中利用原子力顯微鏡(atomic force microscopy, AFM)在細菌近生理狀態下(near-physiological environment)觀察大腸桿菌(Escherichia coli, E. coli) 和線狀噬菌體M13 (filamentous bacteriophage, phage)之交互作用。利用原子力顯微鏡之力曲線(force-distance curve, f-d curve)可以藉由虎克定律(Hooke’s law)以及赫茲模型(Hertz model),去計算出細菌在感染前後之有效彈簧常數 (effective spring constant)以及楊氏係數(Young’s modulus)的變化,發現當細菌被噬菌體感染後有效彈簧常數以及楊氏係數,兩者都有些微的下降。同時利用原子力顯微鏡優異的成像能力,比較在水中大腸桿菌被噬菌體M13感染前感染後的形貌以及粗糙度。比較兩者數據後發現大腸桿菌在被感染後表面變得比較平坦,少了一些凸起物,推測原因是由於細菌表面在被噬菌體感染後,遭受了一些破壞。
    過去研究中提到,當細菌被噬菌體感染後,表面的脂多醣(lipopolysacchride, LPS)會被破壞、釋放。為了證明大腸桿菌被噬菌體感染後表面的LPS會被破壞、釋放,我們使用乙二胺四乙酸二鈉 (ethylenediaminetetraacetates, EDTA)對大腸桿菌做處理,EDTA為二價離子螯合劑,EDTA使得細菌將表面的LPS釋放,將被處理過的大腸桿菌和被感染過的大腸桿菌相比較,可發現不僅在機械性質方面雷同,大腸桿菌膜表面同樣呈現平坦與光滑,因此推斷此一變化的確可能肇因於表面LPS的破壞。除此之外,本研究也利用原子力顯微鏡探針輕壓大腸桿菌並定住,以量取研究當細菌被噬菌體感染後是否會對此壓力有一些不同的現象產生。本研究主要是希望利用原子力顯微鏡探討當細菌被噬菌體感染後之機械性質變化,並希望藉由此一研究能夠發揮原子力顯微鏡在生物研究之優勢,且利用其允許生物在近生理環境下具高解析與可對表面機械性質分析來觀察到其他儀器所不能觀察到的現象。


    論文摘要 I Abstract III 誌謝 V 目錄 VII 表格目錄 XII 第一章 緒論 1 第二章 文獻回顧 3 2.1 噬菌體M13 3 2.2原子力顯微鏡原理以及應用 5 2.2.1 原子力顯微鏡 5 2.2.2 原子力顯微鏡回饋系統 7 2.2.3 原子力顯微鏡操作模式 9 2.2.4 力曲線(force-distance, f-d curve) 10 2.2.5 力曲線(f-d curve)解析 12 2.2.6 力-時間曲線(force-time curve) 12 2.3 原子力顯微鏡在生物及細菌學上應用 14 2.3.1 原子力顯微鏡在溶液環境下的操作 14 2.3.2 利用原子力顯微鏡在生理環境下對細菌成像 17 2.3.3 利用原子力顯微鏡在生物上所做的研究 19 第三章 實驗流程與方法 21 3.1大腸桿菌培養方式以及噬菌體M13感染與確認及EDTA處理 23 3.1.1 大腸桿菌培養方式以及噬菌體之M13感染 23 3.1.2 噬菌體感染確認 24 3.1.3 乙二胺四乙酸二鈉(ethylenediaminetetraacetates, EDTA)處理 25 3.2 大腸桿菌表面水接觸角(contact angle)的量測 26 3.3大腸桿菌固定於基板表面的準備方式以及固定流程 27 3.4 利用原子力顯微鏡成像以及取得力曲線 28 3.5 力曲線的解析 29 3.5.1 有效彈簧常數(effective spring constant)的計算 30 3.5.2 楊氏係數(Young’s modulus)的計算 32 3.5.3 原子力顯微鏡探針彈簧常數量測 34 第四章 實驗結果與討論 35 4.1 大腸桿菌被噬菌體感染率確認 36 4.1.1 溶菌斑測試(plaque assay) 36 4.1.2 免疫螢光測試 37 4.1.3 原子力顯微鏡影像觀察 39 4.2 大腸桿菌表面水接觸角量測 41 4.3 Poly-L-lysine(PLL) 塗佈參數以及細菌固定效果比較 43 4.3.1 PLL塗佈參數 43 4.3.2 PLL對細菌固定之效果 47 4.3.3 大腸桿菌固定於PLL基板平面上在(phosphate buffer saline, PBS)中原子力顯微鏡影像 49 4.4 大腸桿菌有效彈簧常數以及楊氏係數計算 54 4.4.1大腸桿菌有效彈簧常數計算 55 4.4.2大腸桿菌楊氏係數計算 58 4.5力-時間曲線圖 60 4.6 綜合討論 63 第五章 結論 65 第六章 參考文獻 66

    第六章 參考文獻
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