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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/97443完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃筱鈞 | zh_TW |
| dc.contributor.advisor | Hsiao-Chun Huang | en |
| dc.contributor.author | 張祐誠 | zh_TW |
| dc.contributor.author | Yu-Cheng Chang | en |
| dc.date.accessioned | 2025-06-18T16:09:42Z | - |
| dc.date.available | 2025-06-19 | - |
| dc.date.copyright | 2025-06-18 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-05-27 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/97443 | - |
| dc.description.abstract | 人工細胞是透過生物工程技術所設計與構建的功能性顆粒，模擬自然細胞的特定生理功能。這些顆粒通常由生物膜或聚合物膜所構成，內部可包裹具有活性的生物分子，如DNA、RNA、蛋白質等，能夠執行特定的細胞功能，同時避開天然細胞的複雜性。建構一個人工細胞的關鍵方法是無細胞蛋白質合成系統（cell-free protein expression systems 簡稱CFPS），該系統利用細胞萃取或化學合成的方式取得核糖體、tRNA和核苷酸等物質來重現細胞內的轉錄與轉譯過程。當這些 CFPS 組件被封裝於膜結構中，即可形成具備蛋白質合成功能的人工細胞，為探索生命基本機制與合成生物學應用提供了強有力的平台。
在本研究中，我們首先建立了一套光誘導的蛋白質表達系統，該系統利用阿拉伯芥感光蛋白對Cry2PHR/CIB81作為感光元件。這些感光蛋白在藍光照射下發生構型變化，促使Cry2PHR與CIB81形成異源二聚體，進而驅動下游蛋白質表達或定位的變化。為了進一步模擬細胞內的微環境，我們將此光誘導表達系統整合至人工細胞中，透過微流體無細胞平台生成的油包水液滴，這些液滴中封裝了體外轉錄-翻譯試劑，模擬出天然細胞結構和功能的人工細胞室。在這些人工細胞室內，光誘導系統被用來精確調控蛋白質的時空表達，從而促進生物分子的不對稱分佈。這使我們能夠模擬在大腸桿菌這種對稱分裂生物中透過調控蛋白質分佈來建立一個不對稱分裂的系統。這種由簡至繁的研究方法，不僅為分子動態過程的研究提供了新工具，也通過簡化系統克服了活細胞研究的複雜性，推進了合成生物學在人工細胞和合成基因回路構建方面的應用。 | zh_TW |
| dc.description.abstract | Artificial cells, or minimal cells, are engineered particles encapsulating bioactive materials like DNA, RNA, and proteins within a membrane, capable of mimicking cellular functions while bypassing the complexity of native cells. A key approach for constructing these systems is cell-free protein expression systems (CFPS), which utilize essential transcription and translation machinery, such as ribosomes, tRNAs, and nucleotides, sourced from cell extracts or synthesized chemically. Encapsulating CFPS components within membranes enables the creation of functional artificial cells, offering a powerful platform for studying biological processes and advancing synthetic biology applications.
In this study, we first establish a photoinducible protein expression system utilizing photodimerizers composed of Cry2PHR/CIB81 protein pairs derived from Arabidopsis light-sensitive proteins. Under blue light, these proteins undergo conformational changes that trigger heterodimerization, leading to changes in protein expression or localization. To mimic the intracellular environment, we integrate the photoinducible system within artificial cell compartments using a microfluidic cell-free platform. This creates water-in-oil droplets containing IVTT kits, simulating the structure and function of natural cells. Within these artificial compartments, the photoinducible system precisely regulates the spatial and temporal expression of proteins, enabling the creation of asymmetric biomolecule distributions. This allows us to simulate asymmetric division processes in Escherichia coli, which typically exhibits symmetric division, by regulating protein distribution. This bottom-up approach provides a novel method for studying dynamic biological processes in controlled environments and advances synthetic biology applications by overcoming the complexities associated with living cells. It also paves the way for constructing synthetic gene circuits and functional artificial cells in a simplified, artificial setting. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2025-06-18T16:09:42Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2025-06-18T16:09:42Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 致謝 I
摘要 III Abstract IV Contents VI Figure list IX Table list XI Chapter 1. Introduction 1 1.1 Synthetic biology 1 1.2 Asymmetric cell division 1 1.3 Photo-induced expression system 2 1.4 Cell-free protein synthesis system 4 1.5 Microfluidic chip 6 Chapter 2. Materials and Methods 7 2.1 Bacterial strain 7 2.2 Bacterial culture condition 7 2.3 Polymerase chain reaction (PCR) 8 2.4 Biobrick Assembly & Restriction enzyme digestion 11 2.5 Agarose gel electrophoresis 12 2.6 DNA gel extraction 12 2.7 Gibson Assembly 13 2.8 DNA Ligation 14 2.9 Plasmid Transformation 15 2.10 Plasmid DNA Extraction 15 2.11 Fluorescence microscopy 16 2.12 Cell-free protein expression 17 2.13 Microfluidic chip fabrication 20 2.14 Water-in-oil droplet formation 21 Chapter 3. Results and Conclusion 22 3.1 Evaluation of the Microfluidic Cell-Free Platform for Artificial Cell Compartmentalization 22 3.1.1 ELISA-Based Validation of Cell-Free Kits 22 3.1.2 Protein Synthesis in Water-in-Oil Droplets 23 3.1.3 C-Degron Degradation Assay in Cell-Free Kits 24 3.2 Testing the Photoinducible System in Oil Droplets 26 3.2.1 Pre-Test of T7 Recruitment Assay Expression in IVTT Droplets 26 3.2.2 Photoactivation of Full and Split T7-RA in IVTT Droplets 27 3.2.3 Effect of FAD Addition on T7-RA Recruitment in IVTT Droplets 29 3.2.4 Quantification of mCherry Co-localization with GFP Foci 31 3.2.5 Essential Role and Interference of FAD in Recruitment 32 3.2.6 Blue Light-Dependent Activation of T7-RA in IVTT Droplets 33 3.2.7 Optimizing Recruitment Efficiency in IVTT Droplets 35 3.2.8 Conclusion of Photoinducible System in Oil Droplets 36 3.3 Protein Localization in Rod-Shaped Droplets 37 3.3.1 Creating Rod-Shaped Droplets 37 3.3.2 Characterization of DivIVA Protein in Rod-Shaped Droplets 38 3.3.3 Obstacles of Rod-Shaped Droplets Observation 39 3.4 Opto-TEVP: Optogenetic Control of Protein Localization and Function 40 3.4.1 Evaluation of TEV protease activity 41 3.4.2 Validation of the Opto-TEVP System 42 3.4.3 Summary of Opto-TEVP System Evaluation 44 3.5 Conclusion 44 Chapter 4. Discussion and Future Work 48 4.1.1 Localized Recruitment in Rod-Shaped Droplets 48 4.1.2 Optimization of Recruitment Assay 49 4.1.3 Implementing Opto-TEVP in Cell-Free Kit Oil Droplets 52 Figures 54 Reference 81 | - |
| dc.language.iso | en | - |
| dc.subject | 無細胞蛋白質合成系統 | zh_TW |
| dc.subject | 合成生物學 | zh_TW |
| dc.subject | 光誘導系統 | zh_TW |
| dc.subject | 微流體 | zh_TW |
| dc.subject | 油包水液滴 | zh_TW |
| dc.subject | 不對稱分裂 | zh_TW |
| dc.subject | 人工細胞 | zh_TW |
| dc.subject | asymmetric division | en |
| dc.subject | Artificial cells | en |
| dc.subject | Cell-free protein expression systems (CFPS) | en |
| dc.subject | Synthetic biology | en |
| dc.subject | Photoinducible system | en |
| dc.subject | Microfluidic | en |
| dc.subject | Water-in-oil droplets | en |
| dc.title | 在人造細胞中建立一個光誘導系統以對蛋白質進行時間空間上的調控 | zh_TW |
| dc.title | Development of a photoinducible system for spatiotemporal control of proteins in artificial cell compartments | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 涂熊林;吳亘承 | zh_TW |
| dc.contributor.oralexamcommittee | Hsiung-Lin Tu;Hsuan-Chen Wu | en |
| dc.subject.keyword | 人工細胞,無細胞蛋白質合成系統,合成生物學,光誘導系統,微流體,油包水液滴,不對稱分裂, | zh_TW |
| dc.subject.keyword | Artificial cells,Cell-free protein expression systems (CFPS),Synthetic biology,Photoinducible system,Microfluidic,Water-in-oil droplets,asymmetric division, | en |
| dc.relation.page | 86 | - |
| dc.identifier.doi | 10.6342/NTU202500986 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2025-05-27 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | - |
| dc.date.embargo-lift | 2030-05-26 | - |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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