Towards constructing synthetic cells: RNA/RNP evolution and cell-free translational systems in giant liposomes

The emerging field of synthetic biology seems to have a great potential to the development of new biotechnologies and the understanding of self-organizing principle in life. Currently, several synthetic biologists aim to understand the evolution of naturally occurring biological systems through a "bottom-up approach" in contrast to the conventional reductive approach. Several lines of pioneering works have been demonstrated in that behaviors of programmed synthetic networks are predicted in vitro. However, it is crucial to create new molecular "parts" for (re)constructing synthetic genetic networks in artificial cell-like compartments, such as giant liposomes. We focus on RNA and RNP (ribonucleoprotein) that hold promise as new "parts" for synthetic biology. They are constructed with molecular design and an experimental evolution technique. So far, designed self-folding RNAs, RNA (RNP) enzymes, and nanoscale RNA architectures have been successfully constructed by utilizing Watson-Crick base-pairs together with specific RNA-RNA or RNA-protein binding motifs of known defined 3D structures. In addition, we also demonstrated that the cell-sized liposomes can be prepared by using phospholipid-coated micro-droplets, which were generated by emulsification or microfluidic techniques. We encapsulated cell-free translation system (PURE SYSTEM) in newly developed liposomes, and successfully monitored protein expression kinetics within individual liposomes in real time. By combining RNA/protein in vitro evolution techniques with giant liposome-based systems, it may be possible to generate artificial protocells with translational regulatory systems.