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

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。

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.