Minimal model of self-replicating nanocells: a physically embodied information-free scenario

The building of minimal self-reproducing systems with a physical embodiment (generically called protocells) is a great challenge, with implications for both theory and applied sciences. Although the classical view of a living protocell assumes that it includes information-carrying molecules as an essential ingredient, a dividing cell-like structure can be built from a metabolism-container coupled system only. An example of such a system, modelled with dissipative particle dynamics, is presented here. This article demonstrates how a simple coupling between a precursor molecule and surfactant molecules forming micelles can experience a growth-division cycle in a predictable manner, and analyses the influence of crucial parameters on this replication cycle. Implications of these results for origins of cellular life and living technology are outlined.     

Social and ethical checkpoints for bottom-up synthetic biology, or protocells

An alternative to creating novel organisms through the traditional “top-down” approach to synthetic biology involves creating them from the “bottom up” by assembling them from non-living components; the products of this approach are called “protocells.” In this paper we describe how bottom-up and top-down synthetic biology differ, review the current state of protocell research and development, and examine the unique ethical, social, and regulatory issues raised by bottom-up synthetic biology. Protocells have not yet been developed, but many expect this to happen within the next five to ten years. Accordingly, we identify six key checkpoints in protocell development at which particular attention should be given to specific ethical, social and regulatory issues concerning bottom-up synthetic biology, and make ten recommendations for responsible protocell science that are tied to the achievement of these checkpoints.     

An overlooked riddle of life's origins: energy-dependent nucleic acid unzipping

The imposing progress in understanding contemporary life forms on Earth and in manipulating them has not been matched by a comparable progress in understanding the origins of life. This paper argues that a crucial problem of unzipping of the double helix molecule of nucleic acid during its replication has been underrated, if not plainly overlooked, in the theories of life's origin and evolution. A model is presented of how evolution may have solved the problem in its early phase. Similar to several previous models, the model envisages the existence of a protocell, in which osmotic disbalance is being created by accumulation of synthetic products resulting in expansion and division of the protocell. Novel in the model is the presence in the protocell of a double-stranded nucleic acid, with each of its two strands being affixed by its 3'-terminus to the opposite sides of the membrane of a protocell. In the course of the protocell expansion, osmotic force is utilized to pull the two strands longitudinally in opposite directions, unzipping the helix and partitioning the strands between the two daughter protocells. The model is also being used as a background for arguments of why life need operate in cycles. Many formal models of life's origin and evolution have not taken into account the fact that logical possibility does not equal thermodynamic feasibility. A system of self-replication has to consist of both replicators and replicants.     

A Fission-Fusion Origin for Life

To develop a comprehensive cells-first approach to the origin of life, we propose that protocells form spontaneously and that the fission and fusion of these protocells drives the dynamics of their evolution. The fitness criterion for this evolution is taken to be the the stability (conservation) of domains in the protocellular membrane as determined by non-covalent molecular associations between the amphiphiles of the membrane and a subset of the macromolecules in the protocell. In the presence of a source of free energy the macromolecular content of the protocell (co-)evolves as the result of (domain-dependent) membrane-catalysed polymerisation of the prebiotic constituents delivered to the protocell by fusion. The metabolism of the cell therefore (co-)evolves on a rugged fitness landscape. We indicate how domain evolution with the same fitness criterion can potentially give rise to coding. Membrane domains may therefore provide the link between protocells and the RNA/DNA-world.     

Generic Darwinian selection in catalytic protocell assemblies

To satisfy the minimal requirements for life, an information carrying molecular structure must be able to convert resources into building blocks and also be able to adapt to or modify its environment to enhance its own proliferation. Furthermore, new copies of itself must have variable fitness such that evolution is possible. In practical terms, a minimal protocell should be characterized by a strong coupling between its metabolism and genetic subsystem, which is made possible by the container. There is still no general agreement on how such a complex system might have been naturally selected for in a prebiotic environment. However, the historical details are not important for our investigations as they are related to assembling and evolution of protocells in the laboratory. Here, we study three different minimal protocell models of increasing complexity, all of them incorporating the coupling between a 'genetic template', a container and, eventually, a toy metabolism. We show that for any local growth law associated with template self-replication, the overall temporal evolution of all protocell's components follows an exponential growth (efficient or uninhibited autocatalysis). Thus, such a system attains exponential growth through coordinated catalytic growth of its component subsystems, independent of the replication efficiency of the involved subsystems. As exponential growth implies the survival of the fittest in a competitive environment, these results suggest that protocell assemblies could be efficient vehicles in terms of evolving through Darwinian selection.     

Synthetic Turing protocells: vesicle self-reproduction through symmetry-breaking instabilities

The reproduction of a living cell requires a repeatable set of chemical events to be properly coordinated. Such events define a replication cycle, coupling the growth and shape change of the cell membrane with internal metabolic reactions. Although the logic of such process is determined by potentially simple physico-chemical laws, modelling of a full, self-maintained cell cycle is not trivial. Here we present a novel approach to the problem that makes use of so-called symmetry breaking instabilities as the engine of cell growth and division. It is shown that the process occurs as a consequence of the breaking of spatial symmetry and provides a reliable mechanism of vesicle growth and reproduction. Our model opens the possibility of a synthetic protocell lacking information but displaying self-reproduction under a very simple set of chemical reactions.     

Domain protolife

We propose the Thermal Protein First Paradigm (protocell theory) that affirms that first life was cellular. The first cells emerged from molecular (chemical) evolution as protocells (heated amino acids self-order in copolymerization reactions to form thermal proteins which self-organize when in contact with water to form protocells). Metaprotocells are specialized protocells capable of synthesizing ATP (light energy conversion to chemical energy), polypeptides, and polynucleotides. Aggregations of protocells in thermal protein matrices form distinctive morphologies (protocellular networks). Prokaryotic cells emerged from metaprotocells. We classify protocells and metaprotocells as members of the Domain Protolife. We revised the cell theory to include protolife.     

Evolution and self-assembly of protocells

Cells define the minimal building blocks of life. How cellular life emerged and evolved implies to cross the boundary between living and nonliving matter. Here we explore this problem by presenting several relevant components of the whole picture involving chemistry, physics and natural selection. Available evidence suggests that the basic logic of life can be understood and eventually translated into synthetic forms of cellular life. A simple, physically sound model of information-free protocell replication suggests that the basic logic of how to couple metabolism and container can be more relevant than the specific set of parameters used, thus indicating that the emergence of cells might have been easier than we would expect.     

The Origins of Cellular Life

Understanding the origin of cellular life on Earth requires the discovery of plausible pathways for the transition from complex prebiotic chemistry to simple biology, defined as the emergence of chemical assemblies capable of Darwinian evolution. We have proposed that a simple primitive cell, or protocell, would consist of two key components: a protocell membrane that defines a spatially localized compartment, and an informational polymer that allows for the replication and inheritance of functional information. Recent studies of vesicles composed of fatty-acid membranes have shed considerable light on pathways for protocell growth and division, as well as means by which protocells could take up nutrients from their environment. Additional work with genetic polymers has provided insight into the potential for chemical genome replication and compatibility with membrane encapsulation. The integration of a dynamic fatty-acid compartment with robust, generalized genetic polymer replication would yield a laboratory model of a protocell with the potential for classical Darwinian biological evolution, and may help to evaluate potential pathways for the emergence of life on the early Earth. Here we discuss efforts to devise such an integrated protocell model.The emergence of the first cells on the early Earth was the culmination of a long history of prior chemical and geophysical processes. Although recognizing the many gaps in our knowledge of prebiotic chemistry and the early planetary setting in which life emerged, we will assume for the purpose of this review that the requisite chemical building blocks were available, in appropriate environmental settings. This assumption allows us to focus on the various spontaneous and catalyzed assembly processes that could have led to the formation of primitive membranes and early genetic polymers, their coassembly into membrane-encapsulated nucleic acids, and the chemical and physical processes that allowed for their replication. We will discuss recent progress toward the construction of laboratory models of a protocell (Fig. 1), evaluate the remaining steps that must be achieved before a complete protocell model can be constructed, and consider the prospects for the observation of spontaneous Darwinian evolution in laboratory protocells. Although such laboratory studies may not reflect the specific pathways that led to the origin of life on Earth, they are proving to be invaluable in uncovering surprising and unanticipated physical processes that help us to reconstruct plausible pathways and scenarios for the origin of life.Open in a separate windowFigure 1.A simple protocell model based on a replicating vesicle for compartmentalization, and a replicating genome to encode heritable information. A complex environment provides lipids, nucleotides capable of equilibrating across the membrane bilayer, and sources of energy (left), which leads to subsequent replication of the genetic material and growth of the protocell (middle), and finally protocellular division through physical and chemical processes (right). (Reproduced from Mansy et al. 2008 and reprinted with permission from Nature Publishing ©2008.)The term protocell has been used loosely to refer to primitive cells or to the first cells. Here we will use the term protocell to refer specifically to cell-like structures that are spatially delimited by a growing membrane boundary, and that contain replicating genetic information. A protocell differs from a true cell in that the evolution of genomically encoded advantageous functions has not yet occurred. With a genetic material such as RNA (or perhaps one of many other heteropolymers that could provide both heredity and function) and an appropriate environment, the continued replication of a population of protocells will lead inevitably to the spontaneous emergence of new coded functions by the classical mechanism of evolution through variation and natural selection. Once such genomically encoded and therefore heritable functions have evolved, we would consider the system to be a complete, living biological cell, albeit one much simpler than any modern cell (Szostak et al. 2001).     

"Living" under the challenge of information decay: the stochastic corrector model vs. hypercycles

The combined problem of having a large genome size when the accuracy of replication was a limiting factor is probably the most difficult transition to explain at the late stages of RNA world. One solution has been to suggest the existence of a cyclically coupled system of autocatalytic and cross-catalytic molecular mutualists, where each member helps the following member and receives help from the preceding one (i.e., a "hypercycle"). However, such a system is evolutionarily unstable when mutations are taken into account because it lacks individuality. In time, the cooperating networks of genes should have been encapsulated in a cell-like structure. But once the cell was invented, it closely aligned genes' common interests and helped to reduce gene selfishness, so there was no need for hypercycles. A simple package of competing genes, described by the "stochastic corrector model" (SCM), could have provided the solution. Until now, there is no clear demonstration that the proposed mechanisms (compartmentalized hypercycles and the stochastic corrector model) do in fact solve the error threshold problem. Here, we present a Monte Carlo model to test the viability of protocell populations that enclose a hypercyclic (HPC) or a non-hypercyclic (SCM) system when faced with realistic mutation rates before the evolution of efficient enzymic machinery for replication. The numerical results indicate that both systems are efficient information integrators and are able to overcome the danger of information decay in the absence of accurate replication. However, a population of SCM protocells can tolerate higher deleterious mutation rates and reaches an equilibrium mutational load lower than that in a population of HPC protocells.     

The "protocell": a mathematical model of self-maintenance

The concepts of self-generation, autonomous boundary and self-maintenance are explained briefly. The "protocell" is presented as a model of self-maintenance which is based on simple physical mechanisms of diffusion and reaction. The time evolution of the surface of the protocell is taken into account explicitly in the form of a Stefan condition giving rise to a non-linear feedback of the surface motion to the reaction and diffusion processes inside the protocell. The spatio-temporal dynamics are investigated, particularly in the neighbourhood of the stationary states, showing a self-maintaining behaviour under a certain range of nutritional conditions. Under another set of conditions we find an instability leading to a division process so that the population of protocells becomes self-maintaining instead of the single individual. The presented formulation of the protocell model is crucially improved compared with a previous version which required boundary conditions at infinity. The previous version was not strictly self-maintaining since dynamics outside the cell were essential for its behaviour.     

Facilitated diffusion as a method for selective accumulation of materials from the primordial oceans by a lipid-vesicle protocell

A model is proposed for the selective accumulation of amino acids, sugars, nucleotides, cations and protons from the primordial oceans into a lipid vesicle type of protocell. The model is built on facilitated diffusion using simple, primordial, lipid-soluble carriers. The advantages a lipid vesicle protocell would have had over the other potential types of protocells are discussed.     

On a kinetic origin of heredity: minority control in a replicating system with mutually catalytic molecules

As the first step in an investigation of the origin of genetic information, we study how some species of molecules are preserved over cell generations and play an important role in controlling the growth of a cell. We consider a model consisting of protocells. Each protocell contains two mutually catalysing molecule species (X and Y), each of which has catalytically active and inactive types. One of the species Y is assumed to have a slower synthesis speed. Through divisions of the protocells, the system reaches and remains in a state in which there are only a few active Y and almost no inactive Y molecules in most protocells, through the selection of very rare fluctuations. In this state, the active Y molecules are shown to control the behavior of the protocell. The minority molecule species act as the carrier of heredity, due to the relatively discrete nature of its population, in comparison with the majority species which behaves statistically in accordance with the law of large numbers. The minority controlled state may give rise to a selection pressure for mechanisms that ensure the transmission of the minority molecule. Once those mechanisms are in place, the minority molecule becomes the ideal storage device for information to be transmitted across generations, thus giving rise to "genetic information". The relevance of this minority controlled state to evolvability is also discussed.     

Imitational modeling of process of evolution: From organic macromolecules to protocell and animal cell

A dynamic imitational model of initial stages of cell evolution has been developed based on role of environmental calcium concentration. The model is designed from our hypothesis about the medium of the appearance of protocells, which could be potassium water reservoirs rather than sea salt water with its predominance of sodium salts. The necessary elements of the appearance of the protocells served organic molecules, code of their synthesis, and formation of macromolecules under favorable ion concentration in environment: a high K⁺ and Mg²⁺ and a low Na⁺ concentration. The model is based on an assumption that one of the first stages in evolution of life was the appearance in the potassium-magnesium water reservoirs of organic molecules capable for self-replication on the basis of genetic code and formation of protocell with the potassium cytoplasm. The model has demonstrated necessity of formation of cell envelope for development of the protocell. Replacement of the dominant cation in water reservoirs—potassium by sodium—required the appearance of ion-transporting devices in plasma membrane and their participation in adaptation of cells to environment. This stage of evolution was accompanied by the most important morphofunctional event—formation of the plasma membrane instead of cell envelope. The membrane provided the ion asymmetry in the cell (preservation of K⁺ in it) relatively to the sodium external medium for maintaining optimal intracellular medium. In the model system, predecessors of animal cells elaborated mechanism of maintenance of the potassium cytoplasm with the sodium counterion dominating in the environment.     

Phenotypic diversity and chaos in a minimal cell model

Gánti's chemoton model (Gánti, T., 2002. On the early evolution of biological periodicity. Cell. Biol. Int. 26, 729) is considered as an iconic example of a minimal protocell including three key subsystems: membrane, metabolism and information. The three subsystems are connected through stoichiometrical coupling which ensures the existence of a replication cycle for the chemoton. Our detailed exploration of a version of this model indicates that it displays a wide range of complex dynamics, from regularity to chaos. Here, we report the presence of a very rich set of dynamical patterns potentially displayed by a protocell as described by this implementation of a chemoton-like model. The implications for early cellular evolution and synthesis of artificial cells are discussed.     

Origin of sex

The competitive advantage of sex consists in being able to use redundancy to recover lost genetic information while minimizing the cost of redundancy. We show that the major selective forces acting early in evolution lead to RNA protocells in which each protocell contains one genome, since this maximizes the growth rate. However, damages to the RNA which block replication and failure of segregation make it advantageous to fuse periodically with another protocell to restore reproductive ability. This early, simple form of genetic recovery is similar to that occurring in extant segmented single stranded RNA viruses. As duplex DNA became the predominant form of the genetic material, the mechanism of genetic recovery evolved into the more complex process of recombinational repair, found today in a range of species. We thus conclude that sexual reproduction arose early in the evolution of life and has had a continuous evolutionary history. We cite reasons to reject arguments for gaps in the evolutionary sequence of sexual reproduction based on the presumed absence of sex in the cyanobacteria. Concerning the maintenance of the sexual cycle among current organisms, we take care to distinguish between the recombinational and outbreeding aspects of the sexual cycle. We argue that recombination, whether it be in outbreeding organisms, self-fertilizing organisms or automictic parthenogens, is maintained by the advantages of recombinational repair. We also discuss the role of DNA repair in maintaining the outbreeding aspects of the sexual cycle.     

Thermally driven fission of protocells

We propose a simple mechanism for the self-replication of protocells. Our main hypothesis is that the amphiphilic molecules composing the membrane bilayer are synthesized inside the protocell through exothermic chemical reactions. The slow increase of the inner temperature forces the hottest molecules to move from the inner leaflet to the outer leaflet of the bilayer. Because of this outward translocation flow, the outer leaflet grows faster than the inner leaflet. This differential growth increases the mean curvature and amplifies any local shrinking of the protocell until it splits in two. The proposed model, based on mere laws of physics, is a step in the study of the origin of life, as well as a clue for a better understanding of cell proliferation in cancer.     

Sufficient conditions for emergent synchronization in protocell models

In this paper, we study general protocell models aiming to understand the synchronization phenomenon of genetic material and container productions, a necessary condition to ensure sustainable growth in protocells and eventually leading to Darwinian evolution when applied to a population of protocells.Synchronization has been proved to be an emergent property in many relevant protocell models in the class of the so-called surface reaction models, assuming both linear- and non-linear dynamics for the involved chemical reactions. We here extend this analysis by introducing and studying a new class of models where the relevant chemical reactions are assumed to occur inside the protocell, in contrast with the former model where the reaction site was the external surface.While in our previous studies, the replicators were assumed to compete for resources, without any direct interaction among them, we here improve both models by allowing linear interaction between replicators: catalysis and/or inhibition. Extending some techniques previously introduced, we are able to give a quite general analytical answer about the synchronization phenomenon in this more general context. We also report on results of numerical simulations to support the theory, where applicable, and allow the investigation of cases which are not amenable to analytical calculations.     

Coevolution of compositional protocells and their environment

The coevolution of environment and living organisms is well known in nature. Here, it is suggested that similar processes can take place before the onset of life, where protocellular entities, rather than full-fledged living systems, coevolve along with their surroundings. Specifically, it is suggested that the chemical composition of the environment may have governed the chemical repertoire generated within molecular assemblies, compositional protocells, while compounds generated within these protocells altered the chemical composition of the environment. We present an extension of the graded autocatalysis replication domain (GARD) model--the environment exchange polymer GARD (EE-GARD) model. In the new model, molecules, which are formed in a protocellular assembly, may be exported to the environment that surrounds the protocell. Computer simulations of the model using an infinite-sized environment showed that EE-GARD assemblies may assume several distinct quasi-stationary compositions (composomes), similar to the observations in previous variants of the GARD model. A statistical analysis suggested that the repertoire of composomes manifested by the assemblies is independent of time. In simulations with a finite environment, this was not the case. Composomes, which were frequent in the early stages of the simulation disappeared, while others emerged. The change in the frequencies of composomes was found to be correlated with changes induced on the environment by the assembly. The EE-GARD model is the first GARD model to portray a possible time evolution of the composomes repertoire.     

Synthetic biology: an emerging research field in China

Synthetic biology is considered as an emerging research field that will bring new opportunities to biotechnology. There is an expectation that synthetic biology will not only enhance knowledge in basic science, but will also have great potential for practical applications. Synthetic biology is still in an early developmental stage in China. We provide here a review of current Chinese research activities in synthetic biology and its different subfields, such as research on genetic circuits, minimal genomes, chemical synthetic biology, protocells and DNA synthesis, using literature reviews and personal communications with Chinese researchers. To meet the increasing demand for a sustainable development, research on genetic circuits to harness biomass is the most pursed research within Chinese researchers. The environmental concerns are driven force of research on the genetic circuits for bioremediation. The research on minimal genomes is carried on identifying the smallest number of genomes needed for engineering minimal cell factories and research on chemical synthetic biology is focused on artificial proteins and expanded genetic code. The research on protocells is more in combination with the research on molecular-scale motors. The research on DNA synthesis and its commercialisation are also reviewed. As for the perspective on potential future Chinese R&D activities, it will be discussed based on the research capacity and governmental policy.