Paolo Carrara

Compartmentalized reactions as a case of soft-matter biotechnology: synthesis of proteins and nucleic acids inside lipid vesicles

In this mini-review we would like to summarize the recent advances in the field of protein and nucleic acid synthesis inside lipid vesicles (liposomes). This research, which originated within the origin of life community, is now recognized as an example of synthetic biology. Current approaches are based on the convergence of liposome technology and cell-free in vitro technology. In particular, in addition to the classical liposome preparation methods, the new water-in-oil droplet transfer method appears very interesting for progressing in the assembly of these cell-like systems, possibly in combination with microfluidic devices. As an alternative to cell extract, the use of a transcription/translation kit composed of purified components is also presented as a new tool for carrying out protein synthesis inside liposomes. Data presented in the literature are collected and shortly discussed, and the potential relevance of this new soft-matter biotechnology in various research fields is also commented on.

Giant Vesicles “Colonies”: A Model for Primitive Cell Communities

Current research on the origin of life typically focuses on the self‐organisation of molecular components in individual cell‐like compartments, thereby bringing about the emergence of self‐sustaining minimal cells. This is justified by the fact that single cells are the minimal forms of life. No attempts have been made to investigate the cooperative mechanisms that could derive from the assembly of individual compartments. Here we present a novel experimental approach based on vesicles “colonies” as a model of primitive cell communities. Experiments show that several advantages could have favoured primitive cell colonies when compared with isolated primitive cells. In fact there are two novel unexpected features typical of vesicle colonies, namely solute capture and vesicle fusion, which can be seen as the basic physicochemical mechanisms at the origin of life.

Bacterial Division Proteins FtsZ and ZipA Induce Vesicle Shrinkage and Cell Membrane Invagination

Background: Before constriction ZipA anchors FtsZ to the E. coli inner membrane as part of the cell division proto-ring. Results: Dynamic FtsZ polymers shrink ZipA-containing vesicles whereas excess of ZipA invaginates the E. coli membrane destroying the permeability barrier. Conclusion: Constriction forces can be evidenced both in bacteria and in vesicles. Significance: Defined bacterial elements reproduce division functions when assembled in vitro. Permeable vesicles containing the proto-ring anchoring ZipA protein shrink when FtsZ, the main cell division protein, polymerizes in the presence of GTP. Shrinkage, resembling the constriction of the cytoplasmic membrane, occurs at ZipA densities higher than those found in the cell and is modulated by the dynamics of the FtsZ polymer. In vivo, an excess of ZipA generates multilayered membrane inclusions within the cytoplasm and causes the loss of the membrane function as a permeability barrier. Overproduction of ZipA at levels that block septation is accompanied by the displacement of FtsZ and two additional division proteins, FtsA and FtsN, from potential septation sites to clusters that colocalize with ZipA near the membrane. The results show that elementary constriction events mediated by defined elements involved in cell division can be evidenced both in bacteria and in vesicles.

Structure and Enzymatic Properties of Molecular Dendronized Polymer-Enzyme Conjugates and Their Entrapment inside Giant Vesicles

Macromolecular hybrid structures were prepared in which two types of enzymes, horseradish peroxidase (HRP) and bovine erythrocytes Cu,Zn-superoxide dismutase (SOD), were linked to a fluorescently labeled, polycationic, dendronized polymer (denpol). Two homologous denpols of first and second generation were used and compared, and the activities of HRP and SOD of the conjugates were measured in aqueous solution separately and in combination. In the latter case the efficiency of the two enzymes in catalyzing a two-step cascade reaction was evaluated. Both enzymes in the two types of conjugates were highly active and comparable to free enzymes, although the efficiency of the enzymes bound to the second-generation denpol was significantly lower (up to a factor of 2) than the efficiency of HRP and SOD linked to the first-generation denpol. Both conjugates were analyzed by atomic force microscopy (AFM), confirming the expected increase in object size compared to free denpols and demonstrating the presence of enzyme molecules localized along the denpol chains. Finally, giant phospholipid vesicles with diameters of up to about 20 μm containing in their aqueous interior pool a first-generation denpol-HRP conjugate were prepared. The HRP of the entrapped conjugate was shown to remain active toward externally added, membrane-permeable substrates, an important prerequisite for the development of vesicular multienzyme reaction systems.

Approaches to chemical synthetic biology

Synthetic biology is first represented in terms of two complementary aspects, the bio‐engineering one, based on the genetic manipulation of extant microbial forms in order to obtain forms of life which do not exist in nature; and the chemical synthetic biology, an approach mostly based on chemical manipulation for the laboratory synthesis of biological structures that do not exist in nature. The paper is mostly devoted to shortly review chemical synthetic biology projects currently carried out in our laboratory. In particular, we describe: the minimal cell project, then the “Never Born Proteins” and lastly the Never Born RNAs. We describe and critically analyze the main results, emphasizing the possible relevance of chemical synthetic biology for the progress in basic science and biotechnology.

Recent Biophysical Issues About the Preparation of Solute-Filled Lipid Vesicles

Here we report some recent biophysical issues on the preparation of solute-filled lipid vesicles and their relevance to the construction of “synthetic cells.” First, we introduce the “semi-synthetic minimal cells” as the liposome-based cell-like systems, which contain a minimal number of biomolecules required to display simple and complex biological functions. Next, we focus on recent aspects related to the construction of synthetic cells. Emphasis is given to the interplay between the methods of synthetic cell preparation and the physics of solute encapsulation. We briefly introduce the notion of structural and compositional “diversity” in synthetic cell populations.

Approaches to Molecular Communication Between Synthetic Compartments Based on Encapsulated Chemical Oscillators

The use of confined micro-oscillators as paradigmatic model for studying communication and information exchange among network of synthetic cells is rapidly growing in the last years. In this paper we report the first steps of an ongoing investigation on the encapsulation of the Belousov-Zhabotinsky oscillating reaction inside phospholipid vesicles (liposomes). The preparation of liposomes encapsulating water soluble molecules can be efficiently carried out in two steps: 1. confining the solute inside water-in-oil droplet, 2. transformation of droplets into liposomes. Here we have started the investigation of chemical oscillation emerging behavior in these two types of compartments. We firstly show interesting dynamical behavior within emulsion droplets. Next, we assess the influence of additives (sugars in particular, which are necessary for the liposomes production) on the oscillation pattern. Future studies will be devoted to the encapsulation of Belousov-Zhabotinsky reaction within liposomes. The potentiality of our systems for modeling intercellular communication pathways and its applications in the Bio-Chem-ITs context are shortly discussed.

Towards the Engineering of Chemical Communication Between Semi-Synthetic and Natural Cells

The recent advancements in semi-synthetic minimal cell (SSMC) technology pave the way for several interesting scenarios that span from basic scientific advancements to applications in biotechnology. In this short chapter we discuss the relevance of establishing chemical communication between synthetic and natural cells as an important conceptual issue and then discuss it as a new bio/chemical-information and communication technology. To this aim, the state of the art of SSMCs technology is shortly reviewed, and a possible experimental approach based on bacteria quorum sensing mechanisms is proposed and discussed.

Extrinsic stochastic factors (solute partition) in gene expression inside lipid vesicles and lipid-stabilized water-in-oil droplets: a review

Abstract The encapsulation of transcription–translation (TX–TL) machinery inside lipid vesicles and water-in-oil droplets leads to the construction of cytomimetic systems (often called ‘synthetic cells’) for synthetic biology and origins-of-life research. A number of recent reports have shown that protein synthesis inside these microcompartments is highly diverse in terms of rate and amount of synthesized protein. Here, we discuss the role of extrinsic stochastic effects (i.e. solute partition phenomena) as relevant factors contributing to this pattern. We evidence and discuss cases where between-compartment diversity seems to exceed the expected theoretical values. The need of accurate determination of solute content inside individual vesicles or droplets is emphasized, aiming at validating or rejecting the predictions calculated from the standard fluctuations theory. At the same time, we promote the integration of experiments and stochastic modeling to reveal the details of solute encapsulation and intra-compartment reactions.

Recent advancements in synthetic cells research

This paper describes our recent investigations on the construction of synthetic cells. By following a bottom-up synthetic biology approach, we aim at constructing minimal synthetic cells based on the encapsulation of DNA, RNA and proteins within liposomes. We will firstly comment on the physics of solute entrapment inside liposomes, giving emphasis on a remarkable self-concentration effect discovered by us (Luisi et al. 2010, Souza et al. 2011, 2012). Next we will show how it is possible to exploit this phenomenon to reveal the formation of primitive-like, metabolically active cells starting from diluted macromolecular solutions (Stano et al., submitted). In conditions where a protein-synthesis reaction does not proceed at a significant rate, lipid vesicles can entrap all required solutes allowing intraliposome protein production. The second topic deals with the formation of simple, rudimentary primitive cell communities based on giant vesicles (GVs). Oleate-containing GVs associate between each other in the presence of poly-L-arginine to form clusters that might be taken as model of primitive cell communities. Their formation, driven by simple primitive electrostatic interactions bring about a series of distinctive features (stability, enhanced permeability, solute capture, fusion) that might emphasize the role of cooperation in origin of life scenarios, flanking the usual competition issues (Carrara et al., 2012).