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研究生：	孫翊仁 Yi-Jen Sun
論文名稱：	Probing Cell Wall Synthetic Dynamics by Bacterial Flagellar Motor in Escherichia coli
指導教授：	羅健榮 Chien-Jung Lo
口試委員:
學位類別：	碩士 Master
系所名稱：	理學院 - 物理學系 Department of Physics
論文出版年：	2019
畢業學年度：	107
語文別：	英文
論文頁數：	57
中文關鍵詞：	細菌細胞壁、大腸桿菌、細菌鞭毛馬達、螢光顯微鏡
外文關鍵詞：	Bacterial cell wall, Escherichia coli, Bacterial flagellar motor, Fluorescence microscopy
相關次數：	點閱：9 下載：0
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細菌需要不斷分裂來確保種族的延續，由於技術上的困難，在這個動態的生長與分裂過程中，我們對於細菌DNA複製的了解比細胞壁生合成的過程來的詳盡許多。因此在細菌分裂的過程中，有個簡單卻基本的問題我們尚未能解答：「母細菌如何在自身插入新的細胞壁來生成兩個子細胞的細胞壁結構？」
細菌鞭毛馬達的整個結構從細胞內膜一路貫穿到細胞體外，穩固的鑲嵌在細胞壁之中。因此當細菌的細胞壁在生長時，馬達位置的移動只會與細菌插入細胞壁的方式有關。利用這個特性，我們發展了一套新方法，利用細菌鞭毛馬達作為細胞壁上的標記來了解細胞壁插入的機制。
我們將細菌鞭毛馬達標上螢光，並觀察在生長時馬達是如何位移的。利用馬達的位移，我們確認了細菌體中在生長時沒有細胞壁插入的部位以及其範圍。而當細菌在延長自身身長時，馬達在有細胞壁插入的部分會維持在同樣的相對位置，我們由此推論細菌身上任何部位細胞壁插入的速度都是等值的。在細菌進入分裂階段時，位於細菌中心的馬達會逐漸遠離中心，意味著新的細胞隔膜是完全由新的細胞壁材料所構成的。利用以上我們對細胞壁插入機制的了解，我們能利用Bernoulli-shift map來推測馬達在每一代細菌身上的位置。
Bernoulli-shift map平滑化的函數特性告訴我們馬達在細菌表面上的分布是由新生成的馬達所決定的。我們用不同顏色的螢光染劑標記了不同時間點出生的馬達，證明了馬達分布所呈現的凹函數(Concave function)分布是由於馬達傾向於生成在細菌中心所造成的。
有了螢光標記細菌鞭毛馬達這項技術讓我們追蹤馬達的動態，我們對細菌細胞壁生合成機制與穿膜蛋白的分布機制的探討將能有更多的發展性。

Bacterial reproduction is a critical and dynamic life process. Here, we raise a simple yet fundamental question that how does the mother cell remodels the cell wall into two daughter cells? Compare to the DNA replication, we have far less understanding in the mechanism of cell wall remodeling due to the technical difficulties.
In this thesis, we develop a new approach using membrane anchored protein as landmarks to study the cell wall synthesis dynamics. Bacterial flagellar motors (BFM) are membrane protein complexes anchored firmly on the cell wall. Thus BFMs position changes along with cell growth depend solely on the spatiotemporal coordinate of the cell wall insertion.
By tracking fluorophore labeled BFMs in Escherichia coli while cell reproduce, we confirm the existence and determine the size of the cell wall growth inert zone. The normalized axial position of the motor remains constant while cell elongate, indicate a uniform axial cell wall insertion rate. During division, the mid-cell motors will be moved away from cell center indicating that the septum is completely formed by new cell wall material. With the understanding of the cell wall insertion, we built a modified Bernoulli-shift map to predict the position of the motors in each generation once it was formed.
The smoothing property of the Bernoulli-shift map also indicate the motor distribution only determine by the newly born motor. With sequentially labeling motors with different color fluorophore, we are able to distinguish the newly born motors and confirm the concave distribution of the BFM is contributed by the preference of the motors to synthesize in the cell center.
With this new experimental method, we open a new door to study the cell wall dynamics and membrane anchored proteins dynamics.

摘要    i
Abstract    ii
Acknowledgments    iii
Glossary    vi
Acronym    vi
 Introduction    1
1   Bacterial Cell Wall    1
1.1   Function of Bacteria Cell Wall    1
1.2   Types of Bacteria Cell Wall    1
1.3   Structure of Bacteria Cell Wall    2
2   Cell Wall Remodeling    5
2.1   Cell Wall Synthetic Complexes    5
2.2   Cell Wall Insertion    8
3   Bacterial Flagellar Motor (BFM)    11
3.1   Function and Structure    11
3.2   Flagellar Distribution    12
4   Aim of This Work    13
 Material and Method    14
1   Bacterial Strain    14
1.1   Biotinylation    15
2   Fluorescent Dye    17
2.1   Streptavidin-Biotin Interaction    17
3   Growth Condition    19
3.1   Medium/Buffer    19
3.2   Growth Condition    19
4   Technique    20
4.1   Labelling Process    20
4.2   Poly-L-Lysine Slide    20
4.3   Gel Pad    21
5   Apparatus    21
5.1   Microscopy    21
5.2   Phase Contrast    22
5.3   Perfect Focus System (PFS)    23
5.4   Image Acquisition    24
6   Image Analysis    25
6.1   Positioning Center of the Motor    25
6.2   Cell Contour and Midline    25
6.3   Tracking Motor Position on the Cell    26
6.4   BFM Stability on the Cell Envelop    26
6.5   Inert Zone Detection    26
6.6   Cell Wall Insertion in Elongation    27
6.7   Cell Wall Insertion in Division    27
6.8   Predictable BFM Position between generations    27
6.9   Non-uniform BFM Distribution    28
 Results    29
1   BFM Stability on the Cell Envelop    29
2   Inert Zone Detection    30
3   Cell Wall Insertion in Elongation    31
3.1   In Relative Axial Coordinate    31
3.2   Hubble’s Law in cell wall insertion    31
3.3   In Normalized Axial Coordinate    32
4   Cell Wall Insertion in Division    34
4.1   In Normalized Axial Coordinate    34
4.2   Direct Tracking of Newly Born Pole    35
5   Predictable BFM Position between generations    35
5.1   Cell growth and BFM positioning model    35
5.2   Predict BFM Position with Bernoulli Shift Map    36
6   Non-uniform BFM Distribution    38
6.1   Mono-Color Labelling    38
6.2   Simulation    38
6.3   Dual-Color Labelling    40
 Discussion    41
Bibliographies    43
Appendix A    46
Filter Spectrum    46


                                

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