Creating synthetic eukaryotic cells with giant lipid vesicles and microfluidics
Tom Robinson a
a Max Planck Institute of Colloids and Interfaces, Potsdam, Germany
Proceedings of Emerging Investigators in Microfluidics Conference (EIMC)
Online, Spain, 2021 July 20th - 21st
Organizers: Adrian Nightingale, Darius Rackus and Claire Stanley
Invited Speaker, Tom Robinson, presentation 035
Publication date: 5th July 2021

The de novo construction of synthetic cells using non-living components is the approach used by bottom-up synthetic biologists. Building minimal cells and controlling each aspect of their design not only gives us the opportunity to understand real cells and their origins, but also provides alternative routes to novel biotechnologies. Giant unilamellar vesicles (GUVs) are used extensively as scaffolds to construct synthetic cells owing to their compatibility with existing biological components. Microfluidic-based approaches for GUV production show great potential for encapsulating large biomolecules required for mimicking life-like functions (Yandrapalli et al. Micromachines, 2020; Love et al. Angew Chemie, 2020) and recent advances have also given researchers access to high-throughput on-line analysis (Robinson, Adv. Biosyst. 2019; Yandrapalli and Robinson, Lab Chip, 2019; Bhatia, Soft Matter, 2020). Here we present our latest results on how microfluidics can further aid in bottom-up synthetic biology.

First, we will show a microfluidic design that is able to produce surfactant-free pure lipid GUVs in a high-throughput manner (Yandrapalli et al. Commun Chem, 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 (as is commonly overlooked). The design can produce homogenously sized GUVs with tuneable diameters from 10 to 130 µm. Encapsulation is uniform and we show that the membranes are oil-free by multiple tests including measuring the diffusion of lipids via FRAP measurements. 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. doi:10.26434/CHEMRXIV.14593518.V1.). The final synthetic cell comprises three coupled enzymatic reactions, which propagate across three separate compartments in a specific direction due to 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 compartmentalisation has on the biochemical reaction rates.  This work demonstrates the effectiveness of microfluidics for the bottom-up assembly of synthetic cell constructs.

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