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研究生: 楊子瑩
Yang, Tzu-Ying
論文名稱: 運用SOS紅色螢光蛋白微生物感測器偵測DNA損傷藥劑
SOS-red fluorescent protein whole-cell biosensor for the detection of DNA damaging agents
指導教授: 葉怡均
Yeh, Yi-Chun
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2015
畢業學年度: 103
語文別: 中文
論文頁數: 55
中文關鍵詞: SOS紅色螢光蛋白微生物感測器DNA損傷藥劑
英文關鍵詞: SOS-RFP biosensor, DNA damaging agents
論文種類: 學術論文
相關次數: 點閱:136下載:5
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  • 本實驗以大腸桿菌作為SOS紅色螢光蛋白微生物感測器,此生物感測器可以偵測到DNA損傷藥劑便以紅色螢光蛋白作為DNA受損指標。其設計方式為個別以不同的啟動子(PrecA, PsulA, and PdinH)後面接上紅色螢光蛋白序列當作信號基因,設計完的生物感測器分別加入兩種DNA損傷藥劑:絲裂黴素以及萘啶酸,之後分別用了三種不同的儀器進行偵測,此三種儀器分別為: 多功能盤式分析系統(所有細胞一起偵測)、光學顯微鏡、流式細胞儀(對單一細胞進行偵測)。SOS紅色螢光蛋白微生物感測器可以簡單不繁雜的步驟偵測出DNA損傷藥劑。

    E.coli SOS-RFP biosensors which express red fluorescent protein as a signal protein under the control of recA, sulA, and dinH gene promoters in SOS response were constructed for detection of DNA damage and evaluation of DNA damaging agents. Two kinds of DNA damaging agents, which were mitomycin C(MMC), and nalidixic acid(NA), were tested using our E.coli SOS-RFP biosensors cells. We used three techniques to detect responses of biosensors , including microplate reader(detection of whole cells), microscope, and flow cytometry(detection of single cells). The SOS-RFP bacterial biosensors provide a specific and simple method for detecting DNA damaging agents.

    中文摘要 I Abstract II 附圖表目錄 VI 縮寫的全名 XI 第一章 緒論 1 1. 生物感測器 (biosensor) 1 2. DNA損傷與SOS反應 2 2.1 DNA損傷 2 2.2 SOS反應 5 3. SOS紅色螢光蛋白微生物感測器 7 第二章 實驗方法與材料 11 1. 細菌菌株 11 2. 實驗藥品 11 3. 實驗器材 12 4. 實驗方法 12 4.1 質體設計 12 4.2 DNA損傷藥劑配置 18 4.3 偵測微生物感測器 18 第三章 實驗結果與討論 21 1. 螢光效率 21 1.1 加入MMC DNA 損傷藥劑後進行螢光效率偵測 21 1.2 加入NA DNA 損傷藥劑後進行螢光效率偵測 23 2. 時間點追蹤偵測 26 2.1 加入MMC DNA 損傷藥劑後進行時間點追蹤偵測 26 2.2 加入NA DNA 損傷藥劑後進行時間點追蹤偵測 28 3. 光學顯微鏡觀測 30 3.1加入MMC DNA 損傷藥劑後進行顯微鏡偵測 30 3.2加入NA DNA 損傷藥劑後進行顯微鏡偵測 34 4.流式細胞儀偵測 38 4.1 細胞螢光表現分布圖 38 4.2 Coefficient of variation(CV) 45 第四章 結論 50 Reference 52

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