博碩士論文 101222027 詳細資訊




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姓名 林再順(Tsai-shun Lin)  查詢紙本館藏   畢業系所 物理學系
論文名稱
(Investigating Stators Assembly of Flagellar Motors in Escherichia Coli by PALM)
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摘要(中) 這篇論文的主要目的是要探討大腸桿菌(E. coli)細胞膜上的分子馬達固定子(stator)分布的研究。大腸桿菌身長約2微米,牠可以在低雷諾數(Reynolds number)的環境以每秒約20微米的速度前進。大腸桿菌的游泳是透過鞭毛馬達的轉動驅使牠們前進,而鞭毛馬達轉動的方式是透過固定子與轉子(rotor)作用而做旋轉。目前的研究已經知道有兩種型態的固定子,分別為利用鈉離子為能量來源的蛋白複合體(PomA/PomB),以及利用氫離子為能量來源的蛋白複合體(MotA/MotB)。固定子在大腸桿菌分子馬達上的數量至少可以擁有11個,但確切的分布目前仍然尚不清楚,因此我們想要用超高解析顯微鏡PALM,來觀測固定子在寬45奈米分子馬達上的分布。

一般螢光顯微鏡的解析極限大約是200奈米,相對於分子馬達45奈米的尺度,螢光顯微鏡是無法看到個別固定子在分子馬達上的分布。而顯微鏡的解析極限是來自於當兩個點光源同時發光,因為光學繞射的關係使得兩個光源的距離小於200奈米後,強度疊加便無法分辨光源數量與位置。如果只有單一點光源的話,透過影像的擬合分析,點光源的位置可以精準的定位到約一奈米的解析能力。因此,我們藉由可光啟動的螢光蛋白(Photoactivatable GFP, PA-GFP),利用紫外光雷射來控制單一螢光蛋白發光,如此一來我們可以精準的定位單一螢光蛋白位置,同時也可以追蹤這些螢光蛋白的動態行為,透過我們架設的PALM超高解析顯微鏡,我們的系統最好可以達到約10奈米的定位解析能力。

從我們現在超高解析取得的結果顯示,固定子(PomA)在大腸桿菌細胞裡的分布不均勻,兩極的分布會高於細胞的中心。這樣的結果可能是由於固定子取自於Vibrio alginolyticus的鞭毛馬達系統,這種鞭毛馬達在Vibrio身上主要出現於兩極。同時我們亦紀錄了固定子的數量,固定子在大腸桿菌裡的數量經過90分鐘的紀錄,可以記錄到約2000個PomA在過表現細胞約100奈米的厚度裡。我們也做了雙色的螢光實驗,同時標記轉子與固定子的位置。透過PALM影像的重建,我們建立出在200奈米解析度下的固定子在細胞的影像,這樣的影像相似於螢光顯微鏡在螢光蛋白同時發光時會拍攝到的影像。在雙色螢光實驗的影像,固定子分布高的地方與轉子分布高的地方相吻合,確定了固定子分布在馬達轉子周圍,這是第一次藉由光學的方法看到固定子在鞭毛馬達轉子附近的分布情形,無論這些蛋白質在細胞裡是否固定我們都可以看到相似的結果。
摘要(英) This thesis aims to investigate stators assembly of flagella motor in Escherichia coli. E. coli can swim at a speed about 20 µm/s in the liquid at low Reynolds number environment, which is fast compared to the 2 µm cell length. The motor is driven by stators that are nonrotating part of motor. Two kinds of stators have been found, MotA/MotB couples proton flux and PomA/PomB couples sodium ion to the motor rotation. At least 11 stators units surround the motor basal part and work independently. However, the real position of stators around motor basal part is not clear. The diameter of a motor is only 45 nm that is below the resolution of conventional optical microscope. In order to study the stator-assembly and dynamics, we set up a super-resolution microscope to visualize the stator proteins in real motor.

Fluorescent microscopes such as confocal microscopes have resolution limit about 200 nm. It is impossible to resolve the nanostructure of bacteria flagella motor by conventional microscope. The resolution limit is confined at the condition that all the fluorescent probe emission at the same time. If there is only one fluorescent probe emission at one time, it would be possible to localize the central position accurately to only one nanometer. By using photoactivatable green fluorescent protein (PA-GFP) fused stator protein, we can visualize each stator position and monitor the dynamics of stators. By controlling the UV photoactivation laser power to switch on PA-GFP one by one, we can localize each PA-GFP separately at different time to reach 10 nm resolution.
Our current results show the distributions of PomA in cells are not uniform. PomA have higher density in the polar region of cells. It is reasonable because the stator PomA/PomB come from the polar flagella of Vibrio alginolyticus. Total number of PomA is also counted. After 90 minutes recording, there are about 2000 PomA in one cell. Furthermore, in dual color experiment, rotor protein (FliN) and stator proteins (PomA) are colocalized in both fixed and living cells. The dynamical properties of stator proteins are important for the understanding of motor dynamics. Further investigations are needed using our newly developed PALM experimental system.
關鍵字(中) ★ 鞭毛馬達
★ 固定子
★ 大腸桿菌
★ 超高解析顯微鏡
關鍵字(英) ★ Flagellar motor
★ Stator
★ PomA
★ E. coli
★ PALM
論文目次 Introduction 1
1.1 Escherichia coli Background 1
1.2 Flagellar Motor 3
1.3 Stator --- Torque Generating Unit 5
1.4 Torque & Speed 8
1.5 Stator Assembly 10
Apparatus Review 13
2.1 Optical Microscope Principle 13
2.1.1 Phase Contrast 15
2.1.2 Dark Field 16
2.2 Fluorescent Microscopy 17
2.2.1 Emission and Excitation Spectrum 18
2.2.2 Epi-fluorescent Microscope 19
2.2.3 Total Internal Reflection Fluorescence (TIRF) Microscope 20
2.3 Resolution Limit 21
2.3.1 Numerical Aperture (N.A.) 22
2.4 Super-resolution Microscope 23
2.4.1 PALM Background and Principle 24
2.4.2 Photoactivatable Probes-PAGFP 26
2.4.3 PALM Limitation 26
Experimental Strains and Methods 28
3.1 Strains 28
3.2 Super-resolution Microscope Optical Layout 30
3.3 Protocols 31
3.3.1 Preparing Clean Glasses with Base-Bath 31
3.3.2 Preparing Chamber Slide 31
3.3.3 Beads Assay 31
3.3.4 Super-resolution Assay – 20 nm Fluorescent Bead (None Fixed Cell) 32
3.3.5 Super-resolution Assay – 100 nm Gold Bead (None Fixed Cell) 32
3.3.6 Super-resolution Assay – 100 nm Gold Bead (Fixed Cell with Formaldehyde) 33
3.4 Program Analysis Algorithms 33
3.5 Program Parameter Optimization 36
Experimental Results 39
4.1 Strains Growth and Motility Test. 39
4.1.1 Growth Rate. 39
4.1.2 Flagella Rotation Speed Test. 40
4.1.3 Rotor Localization with Tether Cell 43
4.2 Super-resolution Results 44
4.2.1 Data Acquisition 44
4.2.2 Data Analysis 45
4.2.3 Stage Drift Correction 47
4.2.4 PALM Reconstruction of PomA Position 48
4.2.5 Co-localization of Stator and Rotor Proteins. 50
Chapter 5 Conclusion 54
Reference 56
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指導教授 羅健榮(Chien-jung Lo) 審核日期 2014-7-24
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