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研究生:林育青
研究生(外文):Yu Cing Lin
論文名稱:Serratiamarcescens之RssB基因調控組
論文名稱(外文):The RssB regulon of Serratia marcescens
指導教授:賴信志賴信志引用關係
指導教授(外文):H. C. Lai
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
論文頁數:56
中文關鍵詞:細菌表面移行二元系統RssA-RssB基因調控組
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細菌表面移行(swarming)是最原始的生物多細胞行為之一。實驗室之前利用Serratia marcescens swarming做為研究模式來研究其內在機制,發現一套二元系統RssA-RssB可藉由影響S. marcescens swarming啟動的時間點來負向調控swarming,但機制尚未清楚。先前利用pull-down assay初步發現約50個以上不同功能的基因之啟動子可能受磷酸化的RssB (RssB~P)所結合。本研究主要目的為進ㄧ步釐清RssB~P 與這些基因之互動關係及其在swarming中所扮演的角色。我們首先利用電泳遷移率改變實驗(EMSA)確認RssB~P與基因啟動子之間是否為直接結合並初步測試RssB~P與這些DNA片段間的結合力。接著利用反轉錄和即時定量聚合酶連鎖反應探討RssA-RssB在swarming或LB培養液中對於其基因調控組(regulon)的表現所造成的影響及其角色。結果顯示有15個啟動子片段被RssB~P所結合,而在LB培養液中生長的情況下,其中有8個基因在對數生長末期被調控,有10個基因在swarming停滯期中被調控。此項研究可以讓我們初步了解到RssA-RssB訊息傳遞如何調控下游基因調控組,並且對於了解S. marcescens swarming以及多細胞行為發展提供更深入的線索。
Bacterial swarming is a primitive cell differentiation and multicellular behaviour. Using Serratia marcescens as a study model, previously we had identified a pair of two-component system RssA-RssB negatively regulating swarming initiation, especially at the swarming lag phase. However, the underlying mechanism remains unclear. Using pull-down assay, a total of about 50 gene promoters was previously identified to be bound by phosphorylated RssB (RssB~P). To further characterize the interaction between RssB~P and the promoters of these genes, electrophoretic mobility shift assay (EMSA) was performed. At least 15 promoters of genes involved in diverse functions were directly bound by RssB~P with different binding affinity. Reverse transcription and real-time quantitative PCR were used to evaluate effect of RssB~P on transcription level of these genes in bacterial cells grown in different phases. Among the genes whose promoters directly bound by RssB~P, 8 of them were differentially expressed between the parent S. marcescens CH-1 and rssBA deleted cells in late log phase, and 10 genes were differentially expressed before and after swarming initiation. The results of this study imply that RssA-RssB signalling may involve DNA synthesis, virulence, carbon metabolism and chromosome partition to regulate swarming initiation. Taken together, this study offers some clues to explain the role of RssA-RssB signaling during early swarming development.
指導教授推薦書
論文口試委員會審定書
長庚大學授權書 iii
致謝 iv
中文摘要 v
Abstract vi
Contents vii
Figure contents x
Table contents ix
Chapter 1 Introduction 1
1.1 Serratia marcescens 1
1.2 Bacterial swarming behavior 2
1.3 RssA-RssB two-component system signaling 3
1.4 Aims of this study 4
Chapter 2 Materials and methods 6
2.1 Bacterial strains and plasmids 6
2.2 Primers 7
2.3 Enzymes and chemicals 9
2.4 Isolation of plasmid DNA 9
2.5 Preparation of bacterial chromosomal DNA 10
2.6 Extraction of DNA from agarose gels 10
2.7 Restriction enzyme digestion 10
2.8 Ligation reaction 11
2.9 Calcium chloride Transformation 11
2.9.1 Preparation of transformation-competent cells 11
2.9.2 Transformation with plasmid DNA 11
2.10 Blue-white screening for recombinant plasmids 12
2.11 Sequence analysis and databank comparison 12
2.12 Plasmids encoding recombinant proteins 13
2.13 Protein expression 13
2.14 Purification of GST-tagged recombinant proteins 13
2.15 In vitro protein-DNA pull-down assay 14
2.16 Electrophoretic mobility shift assay 15
2.17 Swarming motility assay 16
2.18 RNA extraction 16
2.19 Reverse transcription assay 16
2.20 Real- time quantitative PCR 17
Chapter 3 Results 18
3.1 Experimental design of identifying promoters directly bound by
RssB~P 18
3.2 Fifteen promoter regions were directly bound by RssB~P 19
3.3 Characterization of genes expression under RssAB signaling control 20
Chapter 4 Discussion 22
Reference 25
Appendix I. Standard buffers, solutions 35
Appendix Ⅱ. pBluescript II SK (+/–) vector map and sequence 38
Appendix Ⅲ. Sequences of S. marcescens CH-1 promoter regions bound by GST-RssB~P 39

Fig. 3-1 Experimental design of identifying promoter regions of genes bound by GST-RssB~P. 29
Fig. 3-2 Electrophoretic mobility shift assay of RssB binding regions. 30

Table 2-1 The bacteria strains and plasmids used in this thesis 6
Table 2-2 The primers used in this thesis 7

Table 3-1 Identifying genes expression in the RssB regulon 31
Table 3-2. DNA sequences not bound by GST-RssB~P by EMSA 33
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