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研究生(外文):Tsai, Wen-Tsung
論文名稱(外文):Effect of rpoS on the multicellular behaviour of Serratia marcescence
指導教授(外文):Lai, Hsin-Chih
中文關鍵詞:表面移行多細胞行為sigma factor競爭理論
外文關鍵詞:Serratia marcescenceswarmingrpoSquorum sensingsigma factor competition
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Serratia marcescens 於agar 濃度 0.8 的培養基中,會出現表面移行 (swarming) 的多細胞行為。而表面移行的真正機制並不十分清楚。研究發現表面移行是發生在高細胞密度時。而當細菌生長進入 stationary phase 時,亦是處於高細胞密度狀態,此時 S 會大量產生。S 是一種 alternative sigma factor,能活化一群特定基因的表現,使細菌應付外來的壓力。本篇論文中主要探討 S 是否參與表面移行調控。首先我們從 S. marcescens的phage library 中選殖出完整的 S 基因, rpoS。接著我們將rpoS 在 S. marcescens 體內大量表現,發現 S. marcescens 的活動力和表面移行皆受抑制,但AHL signal的產生並不受影響。我們認為 S 可能是透過 sigma factor 競爭方式來抑制表面移行。

Serratia marcescens shows population surface migration (swarming) behaviour on LB agar plate (0.8 agar). The underlying mechanism why such a phenomenon occurs is not clear. Swarming occurs at high cell density. Bacteria are at high cell density when they grow and enter into stationary phase. The amount of S is overproduced in order to govern the expression of mutiple genes to respond to stress conditions. We hypothesized that S was involved in the regulatory network of swarming. We cloned the S gene---rpoS from S. marcescens by using PCR/phage library. Results showed that while S. marcescens motility and swarming are inhibited in trans by multi-copy rpoSSm, the amout of AHL signal synthesized is not affected. It was hypothesized that S regulates swarming via sigma factor competition with 70.

1.1 Serratia marcescens 的簡介
1.1.1 S. marcescens 的致病力3
1.2 S. marcescens 的分化、表面移行(swarming)及多細胞行
1.2 Quorum sensing system 的簡介4
1.3 Sigma factor 競爭理論6
1.4 S 的簡介8
1.5 S. marcescens 的表面移行調控機制假說11
2.1 細菌株
2.1.1 E. coli strains12
2.1.2 S. marcescens strains13
2.2 載體13
2.3 PCR or Sequence Primers13
2.5.2 抗生素14
2.5.3 化學試劑14
2.5.4 DNA 電泳試劑15
2.5.5 Plaque/Colony hybridyzation reagents15
2.5.6 南方點墨雜交試劑15
2.5.7 質體抽取試劑16
2.5.8 Phage DNA 試劑16
2.5.9 Kits16
2.6 設備17
2.7 其它18
2.8 質體抽取18
2.9 Bacterial chromosome 抽取18
2.10 從agarose gel中純化DNA的方法
2.10.1 DNA在agarose gel上的電泳19
2.10.2 從agarose gel純化出所要的片段19
2.11 重組質體的建立
2.11.1 載體及嵌入DNA的準備19
2.12 Transformation method
2.12.1 Competent cell製備20
2.12.2 質體 Transformation20
2.12.3 篩選20
2.13 從E. coli到S. marcescens的接合生殖(conjugation)20
2.15 Plaque hybridization
3.1 從S. marcescens選殖出rpoS基因26
3.2 將S在細菌體內大量表現來探討S在S. marcescens表
3.3 將rpoS突變掉來探討S在S. marcescens表面移行運動
4.1 S在S. marcescens 表面移行運動中所扮演的角色探討40
4.2 挑選rpoS mutant strain所遇到的困難45

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