Design of a reporter protein for the development of a retroreflection sensing method coupled with a cell-free translation system
- 주제(키워드) cell-free expression system , retroreflection , biosensor , reporter protein , diagnostics
- 주제(DDC) 547
- 발행기관 아주대학교
- 지도교수 유태현
- 발행년도 2023
- 학위수여년월 2023. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000032896
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Cell-free protein synthesis systems allow the study of biological functions without utilizing living organisms. These systems find utility in diverse fields such as synthetic biology, metabolic engineering, and protein engineering. Researchers have combined transcription and translation signal amplification with genetic circuits to enhance the sensitivity of detection in cell-free systems. However, current methods often rely on fluorescent, luminescent or electrochemical signals, necessitating specialized equipment and complex detection procedures. Recently, scientists have explored alternative signals to develop simpler and more convenient detection methods. Retroreflection, the phenomenon where light striking a surface is redirected back to the light source, can be detected using basic optical devices. In this study, we present a method that combines cell-free synthetic biology-based sensing with a straightforward signal detection technique using retroreflective Janus particles (RJPs). Reporter proteins with dual functional parts were designed, where one part interacts with the RJPs and other interacts with the device surface. Consequently, the presence of reporter proteins induces the alignment of RJPs on the surface. Cell-free protein systems have been developed to express a universal reporter protein for multiple targets. This platform technology holds promise for the diagnosis of various diseases.
more목차
1. Introduction 1
1.1 Disease diagnosis 1
1.2 Conventional detection methods of nucleic acid and amino acid 2
1.3 Cell-free protein synthesis 2
1.4 Retroreflection 3
1.5 Aim of the study 4
2. Materials and methods 7
2.1 Plasmids construction 7
2.2 Expression and purification of proteins 7
2.2.1 Universal reporter proteins 7
2.2.2 GST-TAD(P27L) 7
2.2.3 SpyTag003-Z domain-AzF 8
2.3 MST assay 8
2.4 Construction of cell-free protein synthesis systems 9
2.5 Reaction mixture of cell-free translation system 9
2.6 Reaction mixture of Target-assisted synthesis of reporters (TASR) 10
2.6.1 Construction of sensor DNA 10
2.6.2 Annealing of target DNA and sensor DNA 10
2.6.3 Target-assisted synthesis of reporters (TASR) 11
2.7 Reaction mixture of amino acids detection 11
2.8 Conjugation methods 12
2.8.1 Conjugation of SpyTag003-Z domain-AzF / SpyTag003-azide to silica surface of RJPs using branched linker 12
2.8.2 Conjugation of GST-TAD(P27L) to the glass surface using BS3 12
2.8.3 Conjugation of azide-PMIN8A to glass surface using DBCO-sulfo NHS ester 13
2.8.4 Conjugation of azide-PMIN8A to glass surface using branched linker 13
2.9 Procedure of retroreflective sensing of the reporter protein after cell-free protein synthesis 14
2.10 Stability test of SpyTag003-conjugated RJPs 14
3. Results and Discussion 15
3.1 Design of universal reporter protein 15
3.1.1 MDM2-TAD(P27L) pair 15
3.1.2 MDM2-PMI(N8A) pair 16
3.1.3 SpyCatcher/SpyTag system (003 version) 16
3.1.4 Improved universal reporter protein 17
3.1.5 Universal reporter protein functional analysis 20
3.2 Partner peptides conjugation strategy on RJPs and sensing chip surfaces 22
3.3 Pair determination through retroreflective sensing 25
3.4 Universal reporter protein detection through retroreflection after cell-free expression 30
3.5 Optimizations for time in the signal measurement process 30
3.6 Universal reporter protein expression and retroreflective sensing through target DNA recognition 34
3.7 Sensing strategy for amino acids detection 36
3.8 Engineering of universal reporter proteins to increase expression level 38
4. Conclusions 40
5. References 41
6. Abstract in Korean 44
