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論文中文名稱:藉用螢光蛋白融合技術,來觀察鏈黴菌內端粒複製必要蛋白的作用 [以論文名稱查詢館藏系統]
論文英文名稱:To construct fluorescent protein fused Tpc and Tac for in vivo study during replicaton [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
畢業學年度:99
出版年度:100
中文姓名:張鈞博
英文姓名:Jin-Bo Chang
研究生學號:98688013
學位類別:碩士
語文別:中文
口試日期:2011-07-28
論文頁數:133
指導教授中文名:黃志宏
口試委員中文名:陳文盛;陳月茸;楊千金
中文關鍵詞:鏈黴菌線形質體末端蛋白末端伴隨蛋白SCP1
英文關鍵詞:SCP1TpgTapTacTpclinear plasmidStreptomyces
論文中文摘要:鏈黴菌是普遍存在於土壤中的格蘭氏陽性菌,具有線形染色體以及質體,末端會有末端蛋白與DNA 5’端形成共價鍵。鏈黴菌線形DNA複製是由起始點往兩端複製,最後在延滯股(lagging strand)會留下一段無法完成複製的單股DNA,Tap會辨識末端單股DNA,Tap會藉由蛋白質之間的交互作用將末端蛋白與DNA聚合酶帶至單股的DNA,利用末端蛋白作為引子,最後由DNA聚合酶將剩下的區域補齊。SCP1上也有類似Tpg與Tap的兩種蛋白,分別是Tpc與Tac,可能是利用相同的機制結合至末端DNA。
本實驗使用螢光蛋白與Tac以及Tpc在基因層次進行融合,希望藉由觀察螢光蛋白可以了解這些Tac、Tpc在不同細胞分化時期的分佈及運動狀況,最終希望可以藉由Tpc-螢光蛋白來追蹤線形質體在鏈黴菌中的分佈。經由螢光蛋白融合後的質體可以支持質體線形複製,證實其末端蛋白依然具有功能。將質體送至低自體螢光的菌株內觀察,可以得知其末端蛋白的分佈狀態,間接推論出質體的分佈狀況。由實驗結果得知,在鏈黴菌生長過程中紅色螢光(Tpc)與綠色螢光Tac)在不同的細胞分化階段,會有不同的分布狀態,在孢子階段均有紅色螢光(Tpc)與綠色螢光(Tac)的存在。未來可以藉由螢光蛋白的移動來推論鏈黴菌是如何藉由接合生殖將質體傳遞給其他鏈黴菌。
論文英文摘要:Streptomyces are to gram-positive bacteria, they have linear Chromosomes and linear plasmids. Capped by terminal protein covalently bond to the 5’end of DNA. Replication of linear DNA is bidirectional form an internal origin, and replicate the end of DNA the lagging strand cannot be replicated completely, and would leave a single-stranded overhang at the 3’end. In typical Streptomyces chromosomes Tap(terminal associate protein) recognizes the single strand DNA, and lead Tpg(terminal protein) and DNA polymerase to perform patching synthesis using, Tpg as the primer. Linear plasmid SCP1 encodes a different terminal protein, Tpc(Terminal protein of SCP1), and an accompany protein Tac(terminal associate protein of SCP1), both of which are uniqued for end patching in SCP1.
In this study, I used fluorescent protein fused Tpc and Tac, observe the distribution of these protein at different stages of growth and different under the microscope. The geusion protein could support the replication of mini-SCP1 plasmid, prove the fusion. The mini-SCP1 linear plasmid were introduced into a autofluorescent strain S.coelicolor FM145, in Streptomyces cell cycle the fluorescent had different partition. Red(Tpc) and green(Tac) fluorescent can be observed in spore and sporulation, these fluorescent can overlay with DAPI. The fusion gene report system can be use to track the plasmid movement during Streptomyces conjugation.
論文目次:目 錄
中文摘要………………………………………………………………i
英文摘要………………………………………………………………ii
誌謝……………………………………………………………………iv
目錄……………………………………………………………………v
表目錄…………………………………………………………………vii
圖目錄…………………………………………………………………viii
第一章 緒論
1.1鏈黴菌的簡介……………………………………………………1
1.2鏈黴菌的端粒與DNA複製機制…………………………………3
1.3螢光蛋白在鏈黴菌的應用………………………………………5
1.4鏈黴菌FM145的簡介……………………………………………7
1.5本論文的目的……………………………………………………9
第二章 材料與方法
2.1菌種………………………………………………………………10
2.2實驗使用的質體…………………………………………………10
2.3引子設計…………………………………………………………12
2.4藥品、酵素………………………………………………………13
2.5菌種之儲存………………………………………………………13
2.6大腸桿菌的轉型…………………………………………………13
2.7鏈黴菌的轉型……………………………………………………13
2.8鏈黴菌total DNA的分離與純化………………………………14
2.9鏈黴菌環形質體的萃取…………………………………………14
2.10限制酵素及連接酵素的處理…………………………………14
2.11聚合酶連鎖反應,PCR(polymerase chain reaction)………14
2.12在螢光顯微鏡下觀察鏈黴菌……………………………………14
2.13鏈黴菌孢子熱休克的方法………………………………………15
第三章 實驗結果
3.1建構螢光蛋白與Tpc、Tac融合…………………………………16
3.1.1將不同的螢光蛋白基因結合至tac、tpc下游………………16
3.1.2與螢光蛋白融合的Tac、Tpc具有原本的活性………………17
3.1.3建構帶有螢光蛋白的環形質體(pDK704c)……………………18
3.2藉由螢光蛋白觀察Tac、Tpc與Tpg在不同細胞週期的表現狀態………………………………………………………………………32
3.3鏈黴菌孢子萌發過程中的螢光分佈狀態………………………78
3.4藉由螢光蛋白來追蹤質體在孢子中的分佈狀態………………89
第四章 討論…………………………………………………………90
參考文獻………………………………………………………………94
附錄
Appendix 1. Media and buffer……………………………………98
Appendix 2. Competent cell preparation and transformation………………………………………………………102
Appendix 3. Plasmid isolation for E. coli…………………104
Appendix 4. Preparation of Streptomyces protoplast and transfer the plasmid DNA…………………………………………105
Appendix 5. Isolation total DNA for Streptomyces…………107
Appendix 6. 煮沸法萃取鏈黴菌環型質體…………………………108
Appendix 7. Polymerase Chain Reaction (PCR) ………………109
Appendix 8. FM145(pDK704) on agar medium……………………110
Appendix 9. FM145(pDK704c) on agar medium…………………116
Appendix 10. FM145(pLUS892) on agar medium…………………121
Appendix 11. FM145(cy703) on agar medium……………………126
Appendix 12. Spore of FM145(pDK705,Tpc-eGFP)………………132
Appendix 13. Spore of FM145(cy703,Tpg-eGFP)………………133
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論文全文使用權限:同意授權於2014-08-24起公開