Engineering cell-free biosensor platforms for automated and high-throughput screening of cellobiohydrolases and field-applicable arsenic detection = 셀로비오하이드롤라아제의 자동화된 고속 대량 스크리닝과 현장 적용 가능한 비소 검출을 위한 무세포 바이오센서 플랫폼 엔지니어링

목차 (Table of Contents)

• List of Figures 7
• List of Tables 9
• Abbreviations 10
• Abstract 13
• Chapter 1 Research background 16
• 1.1 Introduction 16
• 1.2 Transcription Factor-based biosensor 18
• 1.3 Cell-free protein synthesis system as an enabling platform for synthetic biology 21
• 1.4 Detection of hazardous substances with cell-free TF-based biosensors 26
• 1.5 Enzyme screening with cell-free TF-based biosensor 28
• 1.6 Objectives of this study 29
• Chapter 2 An automated, genetic circuit-based cell-free biosensor for characterization of cellobiohydrolase activity on microcrystalline cellulose 30
• 2.1 Introduction 30
• 2.2 Materials and methods 33
• 2.2.1 Reagents and strains 33
• 2.2.2 Plasmid constructions 35
• 2.2.3 Expression and purification of CelR protein 38
• 2.2.4 Expression and purification of cellulases 39
• 2.2.5 Conventional cellulase activity assay 40
• 2.2.6 Cellulase activity assay using a CB-biosensor with a CFPS system 41
• 2.2.7 Fluorescence signal measurement 42
• 2.3 Results and Discussion 43
• 2.3.1 Developing and optimizing a cell-free CB-biosensor 43
• 2.3.2 Integrating the Echo 525 acoustic liquid handler into the cell-free CB-biosensing reaction 45
• 2.3.3 Characterizing cellobiohydrolase activity using a cell-free CB-biosensor 52
• Chapter 3 Development of a semi-automated workflow for high-throughput screening of cellobiohydrolase activity 58
• 3.1 Introduction 58
• 3.2 Materials and methods 60
• 3.2.1 Reagents and strains 60
• 3.2.2 Plasmid constructions 62
• 3.2.3 Crude extract preparation 65
• 3.2.4 Cell-free protein synthesis 66
• 3.2.5 Semi-automated workflow 67
• 3.2.6 Data Analysis and Statistics 70
• 3.3 Results and Discussion 71
• 3.3.1 Calibration of crude cell extract 71
• 3.3.2 Optimization of reaction conditions for crude extract–based CFPS 73
• 3.3.3 Characterization of a crude extract–based cell-free biosensor for cellobiose detection 75
• 3.3.4 Miniaturization of crude extract–based CFPS system using Echo525 acoustic liquid handler. 79
• 3.3.5 Validation of a high-throughput platform for prototyping cellobiohydrolase activity. 86
• Chapter 4 A field-applicable genetic circuit-based cell-free biosensor for detecting arsenic contamination in seaweed 89
• 4.1 Introduction 89
• 4.2 Materials and methods 93
• 4.2.1 Reagents and strains 93
• 4.2.2 Plasmid constructions 95
• 4.2.3 Expression and purification of the ArsR protein 98
• 4.2.4 Crude extract preparation 99
• 4.2.5 Cell-free protein synthesis 100
• 4.2.6 Real sample test 101
• 4.2.7 Lyophilization and rehydration of CFPS system 102
• 4.3 Results and discussion 103
• 4.3.1 Arsenic biosensor design and optimization 103
• 4.3.2 Specific detection of cell-free arsenic biosensor 109
• 4.3.3 Analysis of real samples using a cell-free arsenic biosensor 111
• 4.3.4 Application of a lyophilized CFPS system for on-site arsenic detection 113
• Conclusion 115
• References 117
• (국문초록) 128