Development of Droplet-Based Microfluidics for Synthetic Biology Applications

Microfluidics combines principles of science and technology, and enables the user to handle, process and manipulate fluids of very small volumes. This technology permits the integration of multiple laboratory applications into one single microfabricated chip, requires minimal manual user intervention and sample consumption, and allows enhanced data analysis speed and precision. Due to these numerous advantages, the potential for this technology to be applied in fundamental biophysical and biomedical research is vast. The major aim of this thesis was to explore the capacities of microfluidics, particularly droplet-based microfluidic technology in the following topics: 1) Mimicry of the immune system cellular environment, with the ultimate goal of programing T cells for adoptive T cell therapy; 2) Bottom-up assembly of minimal synthetic cells. Towards this end, a novel approach to form gold-nanostructured and specifically biofunctionalized water-in-oil droplets was developed. This thesis highlights the advanced properties of nanostructured droplets to serve as 3D antigen presenting cell (APC) surrogates for T-cell stimulation. The combination of flexible biofunctionalization and pliable physical droplet properties work in tandem, providing a flexible and modular system that closely models in situ APC-T cell interactions. The research within this thesis focused also on the dissection of complex cellular sensory machinery implementing an automated droplet-based microfluidic approach. Towards this goal, nanostructured droplets as cell-sized compartments and droplet-based pico-injection technology were used to achieve the bottom-up assembly of the minimal number of proteins required for a “simple synthetic cell.” While the applied methodology has a potential for assembly of a wide range of subcellular functional units, the focus in this thesis was on the reconstitution of the actomyosin cortex. Successful optimization of the biochemical and biophysical conditions within the droplets allowed to achieve precise control over the actin polymerization and actomyosin network organization by their linkage to the droplets periphery. These experimental steps were also necessary to generate signaling events including myosin-driven droplet migration and self-propulsion with reduced molecular complexity compared to living cells