Microfluidic Approaches for the Sequential Bottom-up Assembly of Droplet-based Minimal Synthetic Cells

The formation of lipid-membrane compartments is one of the distinguishing features of eukaryotic cells. Inspired by the complexity of cellular functional modules and compartments, scientists have concentrated on developing synthetic compartments for the bottom-up assembly of cellular functions. This cumulative thesis bridges the gap between the development of droplet-based microfluidic technology, and its application towards the creation of synthetic cells. Towards this end, polymer-based water-in-oil droplets and microfluidic devices were optimized to achieve stable retention of encapsulated biomolecules within the droplet-based compartments. The outcomes of this research provided crucial information for the successful reconstitution of microtubule networks, representing a minimal cytoskeleton model system. Furthermore, in an attempt to combine the properties of biological lipid membranes with the advantages of droplet-based microfluidics, a new compartment system termed droplet-stabilized Giant Unilamellar Vesicles (dsGUVs) was developed. Importantly, the enhanced stability of dsGUVs and the implementation of ’pico-injection’ microfluidic technology enabled the sequential and precise loading of dsGUVs with biomolecules and membrane proteins without compromising their functionality. In order to expand the potential of this technology, a new microfluidic device was developed for the release of the assembled protocells into a physiological environment. Finally, Fluorescence Correlation Spectroscopy (FCS) was reconceptualized as a new analysis tool, allowing for the efficient monitoring of the high-throughput generation process of droplet-based synthetic cells in the kHz-range