Is Research on "Synthetic Cells" Moving to the Next Level?

Abstract

"Synthetic cells" research focuses on the construction of cell-like models by using solute-filled artificial microcompartments with a biomimetic structure. In recent years this bottom-up synthetic biology area has considerably progressed, and the field is currently experiencing a rapid expansion. Here we summarize some technical and theoretical aspects of synthetic cells based on gene expression and other enzymatic reactions inside liposomes, and comment on the most recent trends. Such a tour will be an occasion for asking whether times are ripe for a sort of qualitative jump toward novel SC prototypes: is research on "synthetic cells" moving to a next level?

Keywords: artificial cells; autopoiesis; cell-free protein synthesis; complexity; liposomes; microfluidics; numerical modeling; origins of life; protocells; synthetic biology; synthetic cells.

Figure 1

Synthetic cells made by the encapsulation of chemicals inside artificial compartments. (a) The case of semi-synthetic cells from biochemical components and liposomes. (b) Different types of synthetic cells can be envisaged, depending on the experimental scope. Hybrid systems are also possible. (c) Uses of synthetic cells to basic and applied science.

Figure 2

A schematic representation of the evolution of SC research, with a specific focus on chemical and biochemical reactions inside fatty acid vesicles and lipid vesicles (especially, protein synthesis). Emphasis is given to the development of protein synthesis inside liposomes, as a tool for functionalizing SCs. After the consolidation phase, it seems that in the recent years the sophistication of SC systems is rapidly increasing, possibly leading SCs to a next level, “SCs 2.0”.

Figure 3

Liposome technology and cell-free systems. (a) Giant lipid vesicles are often (but not exclusively) used as compartments for the construction of SCs (image reproduced from [68] published under the CC-BY license). (b) A list of issues in liposome technology. (c) Giant lipid vesicles (and giant polymer vesicles) can be build in highly controlled manner by modern microfluidic technologies (image reproduced from [69] published under the CC-BY license). (d) Cell extracts, typically (but not exclusively) from E. coli, are employed as biomolecular systems for performing in vitro protein synthesis. (e) When encapsulated inside liposomes (or other compartments) they give rise to cell-like systems (i.e., SCs). Image (d) is reproduced, with modifications, from [70] published under the CC-BY license. The reconstituted kit PURE system [71,72], whose composition is known, can be employed in substitution to cell extracts.

Figure 4

Autopoiesis and minimal chemical autopoietic systems. (a) Principles of autopoietic organization. An autopoietic system is defined as a self-bounded chemical system (undergoing continuous transformations) consisting in a confined network of processes that generate and consume the system’s components, including the boundary ones. Note the so-called “structural coupling” with the environment, meaning that the autopoietic system has established (and possibly evolve) its own organization by a dynamical coupling with its environment. (b) Minimal autopoietic chemical systems have been generated in the early 1990s employing micelles, reverse micelles, and vesicles (all based on fatty acids). The typical autopoietic dynamics is shown, consisting in growth and division. Image (b) reproduced from [110] with the permission of Springer-Nature.

Figure 5

Comparison between (a) autopoietic self-reproducing SCs and (b) a robotic representation of self-replicating machines, inspired by the von Neumann self-reproducing automata (original title: “Proposed demonstration of simple robot self-replication”, by NASA Conference Publication 2255 (1982), based on the Advanced Automation for Space Missions NASA/ASEE summer study held at the University of Santa Clara in Santa Clara, California, 23 June–29 August 1980). Image (b) in the public domain [114]. Note that autopoietic chemical systems produce their components from within.

Figure 6

Schematic drawings representing some of the current directions in SC research. (a) Functionalization of the SC membrane-by-membrane proteins or similar components. Note that orientation, shown in (b), becomes an important issue when dealing with vectorial systems as membrane proteins. (c) The nested multicompartment system, or multivesicular vesicle, also known as “vesosome”, is a SC design that allow exploitation of compartmentation, hierarchical levels, chemical gradients across the membranes. (d,e) Assemblies of SCs in two and three dimensions. (f) SCs embedded in biocompatible gel. (g,h) Molecular communication between SCs or between SCs and biological cells.

Figure 7

Interesting approaches for interfacing and exploiting SCs operation in a biological and nanomedicine context. (a) The “nanofactory” proposed by LeDuc and collaborators [170] can recognize a tissue, sense its environment, activate internalized enzymes, and produce a compound of biomedical utility (image reproduced by [170] with the permission of Springer Nature). (b) SC that produces, by gene expression, the Pseudomonas exotoxin A (PEA) and kills breast cancer cells in vivo [178]. (c) SC that senses homoserine lactone (HSL) signals from E. coli and consequently activates its own gene network that ultimately produces a toxin Bac2A, killing the bacterium [179].

Figure 8

Four selected examples of SCs whose behavior is systemic. (a) The α-HL-GFP chimeric protein is produced by TX-TL reactions. It inserts spontaneously in the liposome membrane, forming a pore. Externally present building blocks (and small internal by-products) can pass through the pore (cut-off 3 kDa) allowing a prolonged protein synthesis [4]. (b) DNA is produce inside SCs formed by an ad hoc designed membrane (mixture of several components). Anionic DNA interacts with the cationic membrane and catalysts and facilitates the further binding of membrane precursors, ultimately leading to SC division [14]. (c) Artificial photosynthetic small compartments, including photoactive proteins and ATP synthase have been encapsulated inside a large compartment. Following actinic irradiation with red light, ATP is produced from ADP and inorganic phosphate. The SC uses ATP for polymerising actin into filaments [142]. (d) Upon arabinose activation, SCs of first type produce α-HL, so to form a pore in the membrane. IPTG, which was contained inside these SCs could escape reach a second SC (of different type), which constitutively produces α-HL. Therein, IPTG activates gene expression leading to a final bioluminescence response [17].