There is considerable interest in preparing cell-sized giant unilamellar vesicles from natural or nonnatural amphiphiles because a giant vesicle membrane resembles the self-closed lipid matrix of the plasma membrane of all biological cells. Currently, giant vesicles are applied to investigate certain aspects of biomembranes. Examples include lateral lipid heterogeneities, membrane budding and fission, activities of reconstituted membrane proteins, or membrane permeabilization caused by added chemical compounds. One of the challenging applications of giant vesicles include gene expressions inside the vesicles with the ultimate goal of constructing a dynamic artificial cell-like system that is endowed with all those essential features of living cells that distinguish them from the nonliving form of matter. Although this goal still seems to be far away and currently difficult to reach, it is expected that progress in this and other fields of giant vesicle research strongly depend on whether reliable methods for the reproducible preparation of giant vesicles are available. The key concepts of currently known methods for preparing giant unilamellar vesicles are summarized, and advantages and disadvantages of the main methods are compared and critically discussed.

Following is a synthetic review on the minimal living cell, defined as an artificial or a semi-artificial cell having the minimal and sufficient number of components to be considered alive. We describe concepts and experiments based on these constructions, and we point out that an operational definition of minimal cell does not define a single species, but rather a broad family of interrelated cell-like structures. The relevance of these researches, considering that the minimal cell should also correspond to the early simple cell in the origin of life and early evolution, is also explained. In addition, we present detailed data in relation to minimal genome, with observations cited by several authors who agree on setting the theoretical full-fledged minimal genome to a figure between 200 and 300 genes. However, further theoretical assumptions may significantly reduce this number (i.e. by eliminating ribosomal proteins and by limiting DNA and RNA polymerases to only a few, less specific molecular species). Generally, the experimental approach to minimal cells consists in utilizing liposomes as cell models and in filling them with genes/enzymes corresponding to minimal cellular functions. To date, a few research groups have successfully induced the expression of single proteins, such as the green fluorescence protein, inside liposomes. Here, different approaches are described and compared. Present constructs are still rather far from the minimal cell, and experimental as well as theoretical difficulties opposing further reduction of complexity are discussed. While most of these minimal cell constructions may represent relatively poor imitations of a modern full-fledged cell, further studies will begin precisely from these constructs. In conclusion, we give a brief outline of the next possible steps on the road map to the minimal cell.

Supramolecular chemistry was enriched, about twenty years ago, by the discovery of the self-reproduction of micelles and vesicles. The dynamic aspects and complexity of these systems makes them good models for biological compartments. For example, the self-reproduction of vesicles suggests that the growth in size and number of a vesicle population resembles the pattern of living cells in several aspects, but it take place solely due to physical forces. Several reports demonstrate that reverse micelles, micelles, sub-micrometric and giant vesicles can self-reproduce, generating new particles at the expenses of a suitable precursor. Recently, similar studies are in progress on more complex vesicle-based systems, namely semi-synthetic minimal cells. These are artificial cell-like compartments that are built by filling liposomes with the minimal number of biomolecules, such as DNA, ribosomes, enzymes, etc., in order to construct a living cell in the laboratory. This approach aims to investigate the minimal requirements for molecular systems in order to display some living properties, while it finds relevance in origins of life studies and in synthetic (constructive) biology.

Synthetic biology is an emerging field that aims at constructing artificial biological systems by combining engineering and molecular biology approaches. One of the most ambitious research line concerns the so-called semi-synthetic minimal cells, which are liposome-based system capable of synthesizing the lipids within the liposome surface. This goal can be reached by reconstituting membrane proteins within liposomes and allow them to synthesize lipids. This approach, that can be defined as biochemical, was already reported by us (Schmidli et al. J. Am. Chem. Soc. 113, 8127-8130, 1991). In more advanced models, however, a full reconstruction of the biochemical pathway requires (1) the synthesis of functional membrane enzymes inside liposomes, and (2) the local synthesis of lipids as catalyzed by the in situ synthesized enzymes. Here we show the synthesis and the activity--inside liposomes--of two membrane proteins involved in phospholipids biosynthesis pathway. The proteins, sn-glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT), have been synthesized by using a totally reconstructed cell-free system (PURE system) encapsulated in liposomes. The activities of internally synthesized GPAT and LPAAT were confirmed by detecting the produced lysophosphatidic acid and phosphatidic acid, respectively. Through this procedure, we have implemented the first phase of a design aimed at synthesizing phospholipid membrane from liposome within from within - which corresponds to the autopoietic growth mechanism.

One question in the origin of life is the time at which membrane compartments came into the picture as hosts for the first forms of metabolism. If we assume the proteins and nucleic acids came first, then it is difficult to conceive how all the macromolecular components could have been entrapped at a later time in a single compartment. On the other hand, the hypothesis that metabolism originated from inside the compartment means that we would then have to conceive semipermeable, sophisticated membranes in prebiotic times, which does not appear plausible. With this study, we believe that we can offer a partial solution to this riddle, at the same time opening a new vista on the principles of the entrapment of solute in vesicles. We used cryo-TEM to study the entrapment of the protein ferritin in liposomes. The novel, surprising principle that appears is that when lipid surfaces close up in a proteincontaining solution to form vesicles, the entrapment frequency does not follow the expected Poisson distribution, but tends to assume a power-law behaviour, characterized by many "empty" vesicles (no or very little entrapped solute), and a long decreasing tail with extremely crowded vesicles. Cryo-TEM analysis shows indeed some extremely crowded liposomes adjacent to empty ones. The conclusion is that membrane closure can accumulate a remarkable number of solutes inside some compartments. The possible mechanism and relevance of this extreme local super-concentration effect for the origin of life are discussed.The spontaneous formation of lipid vesicles (liposomes) in an aqueous phase containing one or more solutes produces a heterogeneous population of liposomes in terms of solute content. Such entrapments have generally been studied by averaging techniques, such as batch absorbance or fluorescence, whereas little attention has been devoted to studying individual encapsulation.[1] This is partly due to the technical difficulty of directly counting molecules inside liposomes. The encapsulation of biomacromolecules inside liposomes, on the other hand, is an important issue in origins of life research (protocell models) as well as in recent studies on synthetic cells. [2] A series of recent experiments within our project on the construction of minimal living cells [3] revealed possible deviations from the number of macromolecules expected to be entrapped inside liposomes of diameter d < 200 nm. In particular, with the aim of producing green fluorescent protein (GFP) inside liposomes, we prepared liposomes in the presence of the transcription-translation macromolecular machinery, namely E. coli extracts as well as PURESYSTEM [4] (a cell-free protein synthesis kit containing 36 purified components, t-RNAs, ribosomes, for a total of about 80 different macromolecules). We showed that GFP was synthesised inside liposomes, despite of the fact that the Poisson probability of liposome co-entrapment of about 80 different macromolecules (each at a concentration of 0.1-1 mm) is vanishingly small (~10 À26). In order to explain the obs...