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論文中文名稱:尋找鏈黴菌線型質體SCP1接合傳遞之主要clt [以論文名稱查詢館藏系統]
論文英文名稱:To identify the major cis-acting locus of transfer (clt) of Streptomyces linear plasmid SCP1 [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:化學工程與生物科技系生化與生醫工程碩士班
畢業學年度:104
畢業學期:第一學期
中文姓名:魏佑齊
英文姓名:WEI YU CHI
研究生學號:102688003
學位類別:碩士
語文別:中文
口試日期:2016/01/29
指導教授中文名:黃志宏
口試委員中文名:黃姿雯、陳月茸
中文關鍵詞:鏈黴菌、線型質體SCP1、接合傳遞
英文關鍵詞:Streptomyces, linear plasmid SCP1, conjugation
論文中文摘要:鏈黴菌(Streptomyces)為一種土壤常見微生物,在工業上用來生產抗生素。鏈黴菌的特色為具有線型染色體,並藉由線型或是環型質體進行接合傳遞。接合傳遞機制理解較為清楚的是大腸桿菌環型F質體,藉著滾輪滾動方式進行DNA接合傳遞,但鏈黴菌的線型質體接合傳遞機制還未清楚了解。鏈黴菌的質體上發現有一段接合傳遞序列clt (cis-acting-locus of transfer),這段cis-element提供環型質體或染色體從授予株 (donor) 傳遞至接受株 (recipient) 的能力。所以,了解並找出clt將會對鏈黴菌的接合傳遞機制與功能性有著顯著的幫助。先前實驗室研究中發現SCP1的clt-like接合傳遞位置,但在測試結果在剔除clt-like之後的質體SCP1還是具有接合傳遞的能力,表示其剔除片段為次要clt。依據之前的實驗結果,本實驗將位於SCP1上另一可能clt,長度為12 kb片段繼續尋找clt。為了精確分析位置,將12 kb片段切割成四份且命名為p1~p4。取得p3與p4片段後與在鏈黴菌中不會傳遞的質體進行構築後送入S. lividans 3200 (donor) 與S. lividans TK54 (recipient) 進行接合傳遞效率測試,發現構築後的植體含有p3片段時,微型質體傳遞效率有90%。故推測SCP1 clt可能存在於p3。
論文英文摘要:Streptomyces, a group of Actinobacteria frequently found in soil, is usually applied to produce antibiotics in industry. Streptomyces contains linear chromosome and circular or linear conjugal plasmids. Conjugation mechanism in E. coli is established by F plasmid rolling-circle replication during the process, but the conjugation mechanism in Streptomyces linear replicons is not clear. The element of clt (cis-acting-locus of transfer) has been discovered in Streptomyces that is as a required cis-element for efficiently transferring circular plasmids or chromosome from donor to recipient. Therefore, identification of location of clt in Streptomyces linear plasmids will be helpful to investigate the mechanism of its transfer. Previous studies of our laboratory had found that the clt of SCP1 linear plasmid of Streptomyces was located in internal region and a minor clt with the ability to transfer an un-transferred linear plasmids after it inserted. Unfortunately, this clt did not reduce the transfer frequency of SCP1 after it being knockout. This result indicated there was an another major clt which was identified located in a 12 kb DNA fragment of SCP1. For further narrow down this clt, the DNA fragment was divided into four parts, designed p1~p4. After the p3 and p4 DNA fragments were inserted into un-transferred linear plasmids separately and tested their transfer frequency from S. lividans 3200 (donor) to TK54 (recipient), the p3 fragment displayed 90% transfer frequency of the plasmids. This hints the major clt of SCP1 may locate in p3.
論文目次:第一章 緒論 1
一、接合傳遞的發現 1
二、大腸桿菌接合傳遞的分類 2
三、大腸桿菌的接合傳遞機制 2
四、鏈黴菌簡介 5
五、鏈黴菌的線型染色體與線型質體 7
六、鏈黴菌的接合傳遞 10
七、Tra protein與cis-acting locus of transfer (clt) 12
第二章 材料與方法 16
一、菌種及質體 16
二、藥品及酵素 18
三、緩衝溶液、培養基與抗生素 18
四、菌種保存 18
五、大腸桿菌轉型 19
六、大腸桿菌的質體純化 19
七、鏈黴菌Protoplast製備與轉型 19
八、鏈黴菌DNA純化 19
九、限制酶酵素、T4系列酵素及各種套組使用 20
十、聚合連鎖反應 20
十一、脈衝電場膠體電泳 20
十二、鏈黴菌接合傳遞效率計算 20
第三章 實驗結果 21
一、SCP1上12 kb片段的製備並接入pLUS891質體 21
二、構築pLUS891-p3和pLUS891-p4 24
三、測試帶有SCP1’B部分片段的微型線型質體在3200裡的接合傳遞效率 27
第四章 討論 30
參考文獻 33
附錄 40
附錄I Media and buffer 40
附錄II E. coli Competent cell preparation and transformation 42
附錄III Plasmid isolation from E. coli 45
附錄IV Preparation of Streptomyces protoplast and transformation 46
附錄V Isolation the total DNA of Streptomyces 47
附錄VI Polymerase Chain Reaction 48
附錄VII Pulsed-field gel electrophoresis 49
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