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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | ?進德(Jin-Der Wen) | |
dc.contributor.author | You-Chiun Chang | en |
dc.contributor.author | 張佑群 | zh_TW |
dc.date.accessioned | 2021-06-15T12:50:07Z | - |
dc.date.available | 2016-08-02 | |
dc.date.copyright | 2016-08-02 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-07-21 | |
dc.identifier.citation | 參考文獻
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50639 | - |
dc.description.abstract | 核醣體為一個重要的胞器並存在於所有生物體內,從1953年開始被發現,後來結構陸陸續續地被解出,但是真正的轉譯作用機制尚未明瞭。為此,我們的首要目的是直接觀測核醣體與mRNA之間的交互作用,亦即如何形成複合體、轉譯時核醣體與mRNA的相互運動之結構變化,以及轉譯作用的速率等等。
我們計畫運用單分子螢光共振能量轉移技術 (smFRET)去進行上述的觀測,所以我們必須先製作一個帶有螢光分子的核醣體,在此我們以XL-10大腸桿菌株的rpsE基因做為修飾對象,將ybbR-tag利用基因置換的方式插入內生性rpsE基因3’端,使製造出的核醣體S5蛋白在C端含有ybbR-tag─可以鍵結螢光分子的平台─並透過SFP酵素將螢光分子以共價鍵方式形成穩定鍵結。另外我們也嘗試將SNAP蛋白插入核醣體S5蛋白的C端做出S5-SNAP融合蛋白以期達到在更少步驟下,取得更高螢光標定的目標。我們將修飾過的核醣體以蔗糖梯度方式純化,而mRNA的製備則從人工設計之序列胞外轉錄而來。透過螢光分析我們可以確認螢光分子鍵結在S5修飾蛋白上,利用電泳凝膠遷移分析則可以確認核醣體與mRNA正確形成複合體。冷光素酶活性分析則進一步顯示經修飾過的核醣體具有轉譯活性。最後我們得以用單分子螢光共振能量轉移技術觀測70S核醣體以起始複合體狀態結合mRNA而產生FRET訊號。我們希望藉由進一步的條件優化來改善FRET訊號比例。 | zh_TW |
dc.description.abstract | Ribosomes are important machines for protein synthesis in all organisms. In the past 50 years, structures of the ribosome have been revealed by TEM, X-ray and cryo-EM. However, many aspects of the mechanism of how the ribosome translates mRNA into protein remain unclear. Here we aim to directly probe the interactions between the ribosome and mRNA by single-molecule Förster resonance energy transfer (smFRET). We choose to modify and fluorescently label the ribosomal protein S5 located at the mRNA entrance of the 30S subunit. We insert a ybbR-tag at the C terminus of the endogenous S5 protein (rpsE gene) by genomic replacement. The E. coli mutant grows normally without any apparent defects. Next, we use the SFP synthase to covalently attach Dy-547 or Dy-647 (equivalent to Cy3 or Cy5 respectively) to the ybbR-tag. Alternatively, the SNAP protein is inserted in place of the ybbR-tag, and the S5-SNAP fusion protein is overexpressed from a plasmid as an attempt to achieve better labeling efficiency with a much simpler procedure. After fluorescent labeling, we confirm the presence of the dye on the ribosome by SDS-PAGE and fluorescence imaging. The dye-labeled ribosome appears to be functional as indicated by electrophoretic mobility shift assay (EMSA) and in vitro translation with luciferase reporter assay. Single-molecule FRET signal can be observed from the initiation complex with a dye-labeled mRNA. Further optimization is needed for improvement of the labeling efficiency in order to effectively observe the interactions between the fluorescently labeled ribosome and mRNA. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T12:50:07Z (GMT). No. of bitstreams: 1 ntu-105-R03b43014-1.pdf: 5346888 bytes, checksum: 7b176e6bf7b7825eeeb512377fffe1f7 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 目錄
口試委員會審定書 i 致謝 ii 中文摘要 iii ABSTRACT iv 目錄 v 圖目錄 viii 表目錄 ix 縮寫檢索表 x 第一章 導論 1 1.1 核醣體 1 1.1.1 基本簡介 1 1.1.2 轉譯作用 2 1.2 rpsE基因 3 1.3 SFP synthase 4 1.4 SNAP蛋白 4 1.5 單分子技術 5 1.5.1 基本介紹 5 1.5.2 螢光共振能量轉移 (Föster Resonance Energy Transfer) 6 1.6 研究動機與目的 6 第二章 材料與方法 8 2.1 材料 8 2.1.1 勝任細胞品系 8 2.1.2 質體 8 2.1.3 試劑 8 2.1.4 藥品 9 2.1.5 酵素 12 2.1.6 引子序列 13 2.1.7 溶液 14 2.2 方法 17 2.2.1 質體構築 17 2.2.2 染色體置換實驗 19 2.2.3 大腸桿菌大量培養 22 2.2.4 核醣體純化 23 2.2.5 核醣體重組 32 2.2.6 SFP synthase酵素系統反應 (SFP synthase reaction) 34 2.2.7 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 34 2.2.8 細胞外轉錄作用 ( In vitro transcription) 35 2.2.9 細胞外轉譯作用 (In vitro translation) 36 2.2.10 DNA與RNA黏合反應 (Annealing reaction) 36 2.2.11 電泳凝膠遷移分析 (Electrophoretic Mobility Shift Assay) 37 2.2.12 單分子螢光共振能量轉移實驗 (Single-Molecule FRET) 37 第三章 實驗結果 42 3.1 SFP synthase酵素系統反應 42 3.1.1 優化反應條件 42 3.2 染色體基因置換 43 3.3 改善核醣體純化之方法 43 3.4 核醣體功能檢測 44 3.4.1 核醣體確認 44 3.4.2 電泳凝膠遷移分析 (Electrophoretic Mobility Shift Assay) 45 3.4.3 細胞外轉譯作用活性檢測 (Luciferase reporter assay) 45 3.5 單分子螢光實驗 45 3.5.1 mPKsSD 46 3.5.2 mVEhc1 48 第四章 討論 49 4.1 SFP synthase酵素系統反應之優化條件 49 4.2 基因置換 49 4.3 改善核醣體純化之方法 50 4.4 S5-SNAP融合蛋白 50 4.5 核醣體功能檢測 51 4.5.1電泳凝膠遷移分析 51 4.5.2細胞外轉譯作用活性檢測 51 4.6 單分子螢光實驗 51 4.7 其他 52 4.8 總結 52 參考文獻 54 圖目錄 圖1、SFP synthase酵素系統反應 60 圖2、S5-ybbR質體構築示意圖與序列 62 圖3、S5-SNAP融合蛋白質體示意圖與序列 64 圖4、核醣體純化之折線圖與RNA膠圖 65 圖5、30S重組實驗中所需16S rRNA與TP30之比例運算 66 圖6、冷光素酶活性分析 67 圖7、單分子螢光共振能量轉移實驗材料製備 68 圖8、DNA與RNA黏合設計,mPKsSD 69 圖9、DNA與RNA黏合設計,mVEhc1 70 圖10、大量誘導S5-ybbR蛋白 71 圖11、確認螢光分子鍵結S5-ybbR蛋白 72 圖12、立體障礙影響SFP synthase酵素系統反應 73 圖13、SFP synthase酵素系統反應之優化 74 圖14、設計基因置換之修飾序列 75 圖15、核醣體轉譯起始作用效率 76 圖16、純化核醣體流程示意圖 77 圖17、超高速離心k factor公式與時間、沉降係數關係式 78 圖18、蔗糖濃度黏滯性 79 圖19、核醣體確認 80 圖20、電泳凝膠遷移分析 81 圖21、mPKsSD實驗結果 82 圖22、CoA 547與CoA 647結構 83 圖23、mVEhc1用IC形式之時間軌跡圖 85 圖24、mPKsSD與mVEhc1膠圖 86 表目錄 表1、核醣體純化之各別樣品OD260表 87 表2、SFP synthase酵素反應標定30S螢光相對強度表 88 表3、單分子實驗各別濃度配方 89 表4、個別條件測試簡表 92 | |
dc.language.iso | zh-TW | |
dc.title | 建立單分子研究之基因改造螢光核醣體 | zh_TW |
dc.title | Engineering Fluorescently Labeled Ribosome for Observing mRNA-Ribosome Interactions at Single-Molecule Level | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李弘文(Hung-Wen Li),李以仁(I-Ren Lee) | |
dc.subject.keyword | 核醣體,單分子螢光共振能量轉移,核醣體S5蛋白,rpsE基因,SNAP蛋白, | zh_TW |
dc.subject.keyword | ribosome,single-molecule Forster resonance energy transfer,ribosomal protein S5,rpsE gene,SNAP, | en |
dc.relation.page | 93 | |
dc.identifier.doi | 10.6342/NTU201601113 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2016-07-21 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
顯示於系所單位: | 分子與細胞生物學研究所 |
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