A challenging goal of the synthetic biology is the synthesis of artificial cells from scratch. This research topic sheds insights both on the emergence of life, i.e. the transition between non-living to living matter, and on the preparation of micro-sized confined bio-reactors designed for accomplishing specific tasks, bio-robots.

Lipid vesicles, with a size that can range from few nanometres to tens of microns (Giant Vesicles), are widely used as minimal model cells, also called protocells, since they have an aqueous core enclosed by a lipid bilayer boundary. These compartmentalized systems are useful tools to study membrane protein behaviour, in the hydrophobic domain, and compartmentalized enzyme reactions or any other hydrophilic compound, such as nanoparticles, proteins, DNA and RNA, in the inner aqueous core.

In 2003 Pautot and his co-workers presented the water-in-oil (W∕O) emulsion transfer method suitable to prepare Giant Unilamellar Vesicles (GUVs). All the common preparation methods, such as gentile hydration and electroswelling, don’t allow a well-controlled entrapment of solutes. According to this method water in oil macroemulsion is prepared and after centrifugation, the droplets are converted in GUVs. In this case the water phase of the emulsion contains all the solutes at known concentration. Thanks to this procedure, as I will show in my presentation, a large variety of compounds can be encapsulated such as enzymes, membrane proteins with high molecular weight, small fluorescent molecules, synthetic highly charged polymers, magnetic nanoparticles, etc.

Recently, for the first time, it has been possible to functionalize the lipid membrane of GUVs inserting in the hydrophobic domain an integral membrane protein, the Photosynthetic Reaction Centre (RC), retaining its physiological orientation. In the same way it has been possible to insert several kind of membrane proteins with different properties as well as porins or antibiotics able to create pores through the membrane that can facilitate the transport of small substrates form the external environment to the inner pools.

All these are examples of useful techniques to engineer lipid giant vesicle for designing programmable protocells, able to mimic complex cell behaviour, going step by step towards the artificial cell synthesis.