Bottom-up synthetic biology aims to construct synthetic cells from non-living components. Building biomimetic cells and controlling each aspect of their design not only provides the opportunity to understand real cells and their origins, but also provides alternative routes to novel biotechnologies. Giant unilamellar vesicles (GUVs) are used extensively to construct synthetic cells owing to their compatibility with existing biological components, but traditional methods to form them are limited. Microfluidic-based approaches for GUV production show great potential for encapsulating large biomolecules required for mimicking life-like functions (Yandrapalli et al. Micromachines, 11, 285, 2020; Love et al. Angew Chemie, 59, 5950, 2020). First, we present a microfluidic design that is able to produce surfactant-free pure lipid GUVs in a high-throughput manner (Yandrapalli et al. Commun Chem, 4, 100, 2021). The major advancement is that the lipid membranes are produced in the absence of block co-polymers or surfactants that can affect their biocompatibility - which is commonly overlooked. The design can produce homogenous GUVs with tunable diameters from 10 to 130 µm. Encapsulation is uniform and we show that the membranes are oil-free by measuring the diffusion of lipids via FRAP. Next, we present how we modified this device to encapsulate two sub-populations of nano-sized vesicles for the purpose of establishing enzymatic cascade reactions across membrane-bound compartments, therefore mimicking eukaryotic cells (Shetty et al. ACS Nano, 2021). The final synthetic cell comprises three enzymatic reactions, which propagate across three separate compartments in a specific direction due to size-selective membranes pores. Not only does microfluidics provide a high degree of control over the intra-vesicular conditions such as enzyme concentrations, buffers, and the number of inner compartments, but the monodispersity of our synthetic cells allows us to directly compare the effects that compartmentalization has on the biochemical reaction rates. This work demonstrates the effectiveness of microfluidics for the bottom-up assembly of synthetic cell constructs.

High-throughput production of multi-compartment synthetic eukaryotic cells

ACS Spring 2022

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