Identifying Conditions for Protein Synthesis Inside Giant Vesicles Using Microfluidics toward Standardized Artificial Cell Production

To expand the range of practical applications of artificial cells, it is important to standardize the production process of giant (cell-sized) vesicles that encapsulate reconstituted biochemical reaction systems. For this purpose, a rapidly developing microfluidics-based giant vesicle generation system is a promising approach, similar to the droplet assay systems that are already widespread in the market. In this study, we examined the composition of the solutions used to generate vesicles encapsulating the in vitro transcription-translation (IVTT) system. We show that tuning of the lipid composition and adding poly-(vinyl alcohol) to the outer solution improved the stability of the transition process into the lipid membrane so that protein synthesis proceeded in vesicles. The direct integration of alpha-hemolysin nanopores synthesized in situ was also demonstrated. These protein-synthesizing monodisperse giant vesicles can be prepared by using a simple microfluidic fabrication/operation with a commercial IVTT system.