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研究生： Lita Amalia
Lita Amalia
論文名稱： 基於培養皿演化作為隨機誘變的替代方法
Plate-Based Evolution as an Alternative Approach for Random Mutagenesis
指導教授： 蔡伸隆
Shen-Long Tsai
口試委員: 李振綱
Cheng-Kang Lee
王勝仕
Sheng-Ren Wang
學位類別： 碩士
Master
系所名稱： 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年： 2021
畢業學年度： 109
語文別： 英文
論文頁數： 97
中文關鍵詞： 隨機誘變 、趨化性 、子代噬菌體 、gIII 、半固體培養基
外文關鍵詞： random mutagenesis, chemotaxis, progeny phage, gIII, semi-solid medium
相關次數： 點閱：157  下載：0 
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    • Table of Contents Thesis Recommendation Form.........................................................................................i Qualification Form .......................................................................................................... ii Abstract ........................................................................................................................... iii Acknowledgment...............................................................................................................v List of Figures ............................................................................................................... viii List of Tables.....................................................................................................................x Abbreviation List.............................................................................................................xi Chapter 1 Introduction ....................................................................................................1 1.1. Research Background .........................................................................................1 1.2. Research Objective .............................................................................................2 1.3. Research Content and Proposed Method............................................................3 Chapter 2 Literature Review...........................................................................................5 2.1. Mutagenesis........................................................................................................5 2.2. State-of-the-art Technique in Mutagenesis ........................................................6 2.3. M13 Bacteriophage ............................................................................................7 2.4. PACE (Phage-Assisted Continuous Evolution) ...............................................10 2.5. Chemotaxis and CheZ Gene.............................................................................12 2.6. Mutagenesis Plasmid........................................................................................15 2.7. Green Fluorescent Protein................................................................................16 Chapter 3 Experimental Materials and Methods ........................................................19 3.1. 3.2. 3.3. 3.4. Bacteria and Plasmid........................................................................................20 Material ............................................................................................................21 Equipment and Instrumentation .......................................................................24 Methods............................................................................................................25 3.4.1. Plasmid Extraction (Miniprep) .............................................................25 3.4.2. Polymerase Chain Reaction (PCR).......................................................28 3.4.3. Gel Electrophoresis...............................................................................31 vi 3.4.4. DNA Recovery .....................................................................................33 3.4.5. Restriction Digestion ............................................................................33 3.4.6. DNA Ligation .......................................................................................34 3.4.7. Competent Cell Preparation..................................................................35 3.4.8. Transformation .....................................................................................37 3.4.9. Electrocompetent Cell Preparation .......................................................38 3.4.10. Electroporation/Electro-transformation................................................39 3.4.11. SDS-PAGE ...........................................................................................40 3.4.12. Phage Assembly and Amplification .....................................................44 3.4.13. Bacteriophage Plaque Assay (Phage Titer) ..........................................46 3.4.14. Evolution Plate......................................................................................47 3.4.15. Evolution Plate Recovery .....................................................................50 3.4.16. GFP-Reporter Assay.............................................................................51 Chapter 4 Results and Discussion .................................................................................53 4.1. Plasmid Construction .......................................................................................53 4.1.1. Modification of Accessory Plasmid .....................................................53 4.1.2. Construction of Reporter Plasmid ........................................................55 4.2. Expression of Selection Pressure .....................................................................60 4.3. SDS-PAGE.......................................................................................................64 4.4. Phage Amplification, Titer and Infect Test......................................................66 4.5. Evolution Plate.................................................................................................69 4.5.1. Bacteria chemotaxis .............................................................................70 4.5.2. Mutagenesis..........................................................................................72 Chapter 5 Conclusion .....................................................................................................79 Reference .........................................................................................................................80

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