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研究生:謝沅岏
研究生(外文):Hsieh, Yuan-Wan
論文名稱:探討YtfE對大腸桿菌因外來氧化物質的生長影響和含咪唑及吡啶官能基之陰離子有機接受子的合成與鑑定
論文名稱(外文):The effect of YtfE on the growth of Escherichia coli in the presence of exogenous oxidizing substances and Synthesis and characterization of Imidazole- and pyridine-containing organic receptor for anions
指導教授:洪義盛
指導教授(外文):Horng, Yih-Chern
口試委員:陳建宏江昭毅洪義盛
口試委員(外文):Chen, Chien-HongChiang, Chao-YiHorng, Yih-Chern
口試日期:2017-04-07
學位類別:碩士
校院名稱:國立彰化師範大學
系所名稱:化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:83
中文關鍵詞:鐵硫蛋白基因剔除陰離子有機接受子咪唑及吡啶官能基
外文關鍵詞:iron-sulfur proteinsgene knockoutanionorganic receptorImidazole- and pyridine-
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Part I : 含鐵和鐵硫基團的鐵硫蛋白(iron-sulfur proteins)參與生物體內許多重要的生化反應,包括氧分子運輸和利用、中間代謝、檸檬酸循環、電子傳遞鏈、基因轉錄及DNA修補等代謝途徑,但易受到外界的不同壓力刺激而失去其活性,進而影響生物生長。近年來發現大腸桿菌中的YtfE蛋白,可能幫助修復在氧化壓力下被破壞的鐵硫結構蛋白中扮演重要角色。
研究中首先生產出大腸桿菌染色體ytfE基因剔除的突變株,主要是利用基因同源重組原理,使導入的基因片段與宿主染色體上的目標序列進行同源重組,使大腸桿菌染色體上的目標基因產生改變,進而失去其活性。另一方面為比較原始菌種與剔除突變後的菌株生長表現,嘗試以不同的生長環境條件比較兩者間的差異,並找出YtfE存在與否,對其生理生長上產生的影響。
根據實驗結果,YtfE在具有雙氧水培養環境下,確實有幫助細菌回復正常生長的效果,但將YtfE以質體構築後重新送回菌株內後,生長反而出現停滯現象。而同樣藉由一氧化氮和硝酸根離子下等含氮自由基的環境下培養,則皆無明顯的生長差異表現。
Part II :另外陰離子在日常生活環境中扮演著很重要的角色,例如硫酸根的存在會影響核廢料處理過程中的玻璃化問題。因此,合成對特定陰離子有辨識性的接受子,是近年化學家們關心的議題之一。這類對於陰離子有辨識性的接受子,大都具有提供氫鍵作用力的位置,或是以其他非共價鍵作用力抓取目標陰離子。
我們成功合成出一個具有七個氮並有三個吡啶和兩個咪唑[1,5-a]吡啶官能基團之陰離子有機接受子,利用1H NMR和UV-Vis螢光光譜的偵測方式,添加不同陰離子後對接受子的影響,在對硫酸根離子在甲醇溶液下,其NMR和UV-Vis螢光顯示作用最為明顯,之後做滴定實驗以Stern-Volmer Plot和job’s plot計算與硫酸根離子間結合的比例為1 : 1和結合常數是7.05×104 M-1。
Part I :
Iron-sulfur (Fe-S) proteins containing iron-sulfur clusters are involved in many important biochemical reactions in living organism. The loss of activities of these proteins are easily found in the cells under oxidative stress, and affects the biological growth of cells. In recent years, YtfE proteins are proposed to play an important role in repairing iron-sulfur proteins that are damaged by oxidative stress. To find out whether the existence of YtfE proteins affects cell growth under oxidative stress conditions, the ytfE gene knockout strain of E. coli was produced by using the principle of homologous gene recombination. The cell growth profiles of the original strain and the knockout strain with various conditions were compared. We found that YtfE had the effect of helping bacteria cells to return to the normal growth in culture with hydrogen peroxide, but not nitric oxide.

Part II :
Anions play an important role in our daily life. Thus, synthetic organic receptors for specific anion is one of the topics of concern to chemists in recent years. We synthesized successfully an organic receptor containing two imidazol [1,5-a] pyridine functionalities. By using 1H NMR, UV-Vis, and fluorescence spectroscopies, we found that this receptor specifically selects and binds sulfate anions in DMSO and methanol solutions. Through the Stern-Volmer and the Job plots based on the titration experiments, the optimal ratio of 1: 1 and the binding constant of 7.05 × 104 M-1 were determined.
第一章、 緒論 1
Part I 1
第一節 前言 1
第二節 大腸桿菌介紹 1
第三節 自由基(free radical)介紹 2
第四節 鐵硫簇( iron-sulfur clusters )介紹 3
第五節 YtfE蛋白介紹與其相關的修復作用 4
第六節 Part I整理與工作內容概述 5
Part II 7
第七節 超分子化學 7
第八節 陰離子介紹 8
第九節 含氫鍵提供者的接受子及其對應陰離子的辨識 9
1-9-1 含imidazole官能基的三腳架狀配位基 9
第十節 螢光發光的原理 11
1-10-1 誘導電荷轉移(photo-induced charge transfer,PCT) 11
1-10-2 單體-激發態雙體(Monomer-Excimer) 12
1-10-3 C=N的異構(C=N isomerization) 12
第十一節 Part II工作內容概述 14
第二章、 實驗部分 15
Part I 15
第一節 基本原理 15
2-1-1 Red同源重組系統 15
2-1-2 聚合酶連鎖反應(Polymerase chain reaction,PCR)原理 16
2-1-3 批次培養(batch culture)之細胞生長模式 16
2-1-4 菌體倍增時間換算公式 16
2-1-5 DNA 濃度測量 17
第二節 菌種儲存與培養 18
2-2-1 實驗菌種 18
2-2-2 菌種培養 18
2-2-3 培養基製備 18
2-2-4 使用抗生素 19
第三節 DNA純化方法 19
2-3-1 質體萃取 19
2-3-2 染色體DNA萃取 20
2-3-3 瓊脂凝膠中DNA萃取 20
2-3-4 DNA純化 21
第四節 剪切反應、連接反應、凝膠電泳法 21
2-4-1 剪切反應(Digestion) 21
2-4-2 連接反應(Ligation) 21
2-4-3 膠體電泳(Agarose Gel Electrophoresis) 22
2-4-4 DNA定序 22
第五節 勝任細胞製備方式 22
2-5-1 化學法勝任細胞製備 22
2-5-2 電擊穿透法(Electroporation)的勝任細胞製備 23
第六節 轉型作用(Transformation) 23
2-6-1 以化學法製備勝任細胞的轉型,熱休克法 23
2-6-2 以電擊穿透法製備勝任細胞的轉型 24
第七節 聚合酵素連鎖反應(Polymerase Chain Reaction) 24
第八節 建立 E. coli ATCC23716 之YtfE蛋白剔除突變菌株 24
2-8-1 勝任細胞之製備 24
2-8-2 pRedET質體之轉型作用 25
2-8-3 建立一段線型PCR產物 25
2-8-4 進行染色體上之ytfE基因的取代 25
2-8-5 PCR反應確認取代位置 26
第九節 質體建構(plasmid construction ) 26
第十節 定點突變(site-directed mutagenesis) 26
第十一節 一般生長曲線量測 27
第十二節 無氧環境下不同硝酸鹽對於大腸桿菌YtfE突變株的生長影響 27
第十三節 在雙氧水的抗氧化壓力能力測試 27
第十四節 一氧化氮飽和水的製備 27
第十五節 在一氧化氮下的抗氧化壓力能力測試 28
Part II 29
第十六節 一般實驗 29
第十七節 物理性質測量 30
第十八節 含七個氮接受子的合成 31
2-18-1 合成Dimethyl pyridine-2,6-dicarboxylate (1) 31
2-18-2 合成pyridine-2,6-dimethanol(2) 31
2-18-3 合成2,6-bis(bromomethyl)pyridine(3) 31
2-18-4 合成 2,6-bis(azidomethyl)pyridine(4) 32
2-18-5 合成 (6-(aminomethyl)pyridine-2-yl)methanamine(5) 32
2-18-6 合成化合物六,以6[OH]2表示 33
第三章、 結果與討論 34
Part I 34
第一節 製作 E. coli ATCC23716 ytfE 剔除突變株 34
第二節 pytfE質體構築 36
第三節 以不同受體在厭氧環境下大腸桿菌菌株的生長差異比較 37
第四節 測試以雙氧水為來源的抗氧化壓力能力 43
第五節 測試以一氧化氮為來源的抗氧化壓力能力 45
Part II 47
第六節 含7個氮的接受子化合物6[OH]2合成 47
3-6-1 含七個氮的接受子6[OH]2合成 48
3-6-2 6與不同陰離子的X-ray晶體結構鑑定與分析 50
第七節 6[OH]2對不同陰離子的篩選結果 52
第八節 6[OH]2與硫酸根的螢光滴定實驗和Job Plot實驗 55
第九節 不同陰離子與化合物6紅外線光譜分析 62
第四章、 結論與未來展望 63
第五章、 參考文獻 64
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