Synthetic membranes in microfluidic interfaces

This thesis explores the development of microfluidic technology for generating and manipulating micro-sized vesicles with the incorporation of specific membrane proteins as artificial cellular systems to mimic natural existing cells. Synthetic biology (SynBio) is an emerging area of research concerned with the application of engineering methods to the creation of new biological processes and constructs. Understanding the working principle of living cellular system is one of significant issue for scientists working in this field. Cells are known as the basic unit of life: creating model synthetic analogues offers opportunities for us to deepen our insights of complex interaction and to understand features and functions of the living cells. Microfluidic technologies have provided the capabilities of compartmentalisation, monodispersity and high-throughput generation for engineering architectures resembling cell-like structures. In vitro transcription and translation (IVTT) enables the expression of specific proteins of interest within synthetic cells via encapsulation of cell-free protein expression solution has demonstrated artificial cells with the capability of containing the process of central dogma of molecular biology. The thesis investigates the building of synthetic cell-like constructs by microfluidics. The first area of investigation focusses on the fabrication of lipid/polymer vesicles transformed from ultra-thin shell double emulsions, which were prepared using microfluidics. To bring the biological function into both vesicle-based synthetic chassis, a fluorescent protein and a pore-forming membrane protein were in vitro expressed in the artificial cell chassis. The second area of study centres on the viscosity analysis of artificial cell membranes using a combination of molecular rotors and the fluorescence lifetime imaging microscopy. The membrane viscosity plays a crucial role in membrane proteins insertion that influences the cell function regulation through functional biomembranes. The alteration of lifetime of the molecular rotors trapped in the artificial membranes reports the viscosity changes in the membrane environment induced by the dewetting process. Comparisons of viscosity values over time between lipid vesicle templated by thin-shell double emulsions with GUVs produced by an oil-free method (electroformation) offers the ability of measuring the amount of oil phase (organic solvent mixture) in artificial cell membranes. In the final chapter, the research detailed the construction of lipid bilayers with asymmetric arrangement used as more complex artificial cell models compared with most of synthetic cells with symmetric composition in their bilayers. A vesicle with hybrid asymmetric bilayer is also fabricated in same microfluidic fashion where phospholipid deposits on the inner-leaflet and block copolymer coats the outer monolayer. Taken together, the work presented in this thesis shows the potential to exploit the microfluidic construction of a functioning synthetic cell from individual molecular components, which could advance new application areas in biotechnology and health. Further developments in this research will aim to develop microfluidic technologies for: (i) physically investigating cell division process using lipid vesicles as cell models; and (ii) producing complex multicompartmental systems for the use of mimicking natural cells. The asymmetric bilayers will be studied for their influences on the integration of transmembrane proteins.