This project aimed to construct suitable compartments for synthetic cells. Such compartments should be biocompatible and be able to support a range of complex (bio)chemical processes. One of the most promising compartments are liposomes. Liposomes very much resemble the cell membrane and are composed of the same lipids. However, the formation of liposomes is typically uncontrolled and yields a highly polydisperse mixture. Importantly, the content of liposomes cannot be chosen, as it will be the same as the surrounding fluid. This complicates the study of reactions inside liposomes, as it is very difficult to guarantee that no reaction takes place in the exterior. The objective of this study was to find new methods for liposome formation. Using a microfluidic device, we were able to produce double emulsions (water in oil in water, W/O/W) which are precursors to liposomes. They key step is that the lipids stabilizing the oil/water interfaces must come together to form a lipid bilayer. To this end, the oil has to be removed. Typically, this will result in a large excess of lipid drying into a thin shell. However, by carefully controlled the kinetics of the evaporation of oil, and the concomitant changes in surface tension, we were able to dewet perfect liposomes from oil droplets. After establishing this technology, and expanding it to form liposomes-in-liposomes, hierarchical liposomes and vesosomes (i.e. all kind of architectures where different liposomes are placed in close proximity) we set about using these liposomes as containers for biochemical reactions. We established that it is possible to carry out enzymatic reactions as well as gene expression inside liposomes. By perforating the lipid bilayers using pore-forming proteins, we were able to extend reactions by feeding additional reagents from the outside. And finally, we were able to shrink and swell liposomes, thereby inducing phase transitions inside.
This project has contributed to efforts to build a synthetic cell. This is an effort that touches fundamental questions about life: what is life, and where does it come from. To answer these questions we must understand how life is organized, how reactions in tiny volumes are balanced and how we create functional systems out of relatively simple building blocks.
