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論文中文名稱:找尋鏈黴菌線型質體SLP2接合傳遞的主要cis-acting elements [以論文名稱查詢館藏系統]
論文英文名稱:Identification of crucial cis-acting elements on Streptomyces linear plasmid SLP2
for conjugal transfer [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:鄭宇良
英文姓名:Yu-Liang Zheng
研究生學號:98688009
學位類別:碩士
語文別:中文
口試日期:2011-07-28
論文頁數:107
指導教授中文名:黃志宏
口試委員中文名:陳文盛;楊千金;陳月茸
中文關鍵詞:鏈黴菌SLP2同源重組接合傳遞的起始點
英文關鍵詞:Streptomyceshomologous recombinationSLP2oriT
論文中文摘要:SLP2是Streptomyces lividans內的50 kb線型質體可進行接合傳遞,而接合傳遞現象以大腸桿菌F plasmid環型質體的研究較透徹,屬於單股DNA進行單向傳遞,但其作用機轉無法用於解釋鏈黴菌線型質體的傳遞現象。本實驗室希望藉由找出SLP2的clt區塊來知道複製的起始點 (oriT) ,先前實驗藉由質體接有2 kb的clt片段就能夠將無法被傳遞的線型質體傳遞到接受株裡面,顯示其接合傳遞主要的DNA片段位於在這2 kb的範圍。但未證實在進行接合傳遞的起始點 (oriT) 在此2 kb片段,所以此片段暫時稱為clt (cis-acting locus of transfer)。
本論文繼續先前的研究,從這2 kb範圍繼續縮小clt的位置,將此片段分成幾個小片段接到質體上,分析質體在接有哪個小片段時可以將原本不能傳遞的質體在線型質體SLP2接合傳遞時順勢將質體傳遞到接受株。分析實驗結果,主要的clt是一段約450 bp的片段。在許多研究中,tra基因被認為是幫助質體傳遞的主要基因,但在鏈黴菌中過度產生Tra protein時會具有毒殺性的現象,所以在部份的鏈黴菌內都有發現所謂的kil-kor system,藉由重組質體只含有SLP2 tra基因而在沒有SLP2的背景之下,發現其鏈黴菌依然可以存活,推測此鏈黴菌可能不屬於kil-kor system。
論文英文摘要:Streptomyces lividans have a 50 kb SLP2 linear plasmid. The linear plasmid can be transfer to other Streptomyces during the conjugation. The current established mechanism of conjugation is mostly based on circular F plasmid in E. coli system. But that one-way, single-strand DNA transfer mechanism couldn’t be applied to linear plasmid conjugal transfer of Streptomyces. Our lab target on locating the replication origin (oriT) by identifying the clt (cis-acting locus of transfer) region of S. lividans SLP2 plasmid. Our previous study showed that non-transferrable linear plasmid carring 2 kb SLP2 clt fragment resulted in successful transfer to recipient. The data showed that the oriT is located in the 2 kb region. But yet to confirmed the DNA transfer during conjugation start from this region so this fragment is temporarily named clt.
Thesis focuses narrow down the 2 kb clt containing region. The non-transferrable homologous plasmids with different fragment of the 2 kb clt containing region and
iv
observing with plasmid with small clt fragment that can be transferred to the recipient under linear plasmid SLP2 conjugation background. By this strategy, I found the clt is a fragment of length about 450 bp. In many studies, tra had been reported as a main factor that helps plasmid transfer. However, overexpression of tra resulted in cytotoxicity. Thus, kil-kor system can be found in most of the Streptomyces to control tra expression. In this study, recombinant plasmid with tra not causing observable cell death. This result suggests that SLP2 tra might under regulation of gemonic kil-kor system or the regulatory mechanism is different from the general kil-kor/tra system.
論文目次:中文摘要……………………………………………………………………i
英文摘要……………………………………………………………………ii
目錄…………………………………………………………………………v
表目錄………………………………………………………………………vii
圖目錄………………………………………………………………………viii
第一章 緒論………………………………………………………………1
第二章 材料與方法………………………………………………………12
第三章 實驗結果
3.1將2 kb大小的conjugal cis-acting element繼續縮短其範圍…23
3.2將原本2 kb的cis-acting locus of transfer細分成更小片段
來測詴……………………………………………………………35
3.3檢測f片段長度延伸的接合傳遞能力…………………………51
3.4刪除SLP2 19.4~19.6 kb的DNA序列………………………….69
3-5分析突變的S. lividans 1326之接合傳遞能力…………………75
第四章 討論………………………………………………………………78
參考文獻……………………………………………………………………84
附錄
Appendix A. Media and buffer…………………………………………91
Appendix B. Competent cell preparation and transformation…………94
Appendix C. Plasmid isolation for E. coli……………………………97
Appendix D. Preparation of Streptomyces protoplast and transfer the
plasmid DNA……………………………………………98
Appendix E. Isolation of total DNA for Streptomyces…………………99
vii
Appendix F. Polymerase Chain Reaction (PCR)………………………100
Appendix G. Pulsed-field gel electrophoresis (PFGE) ………………101
Appendix H. Southern hybridization. ………………………………104
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