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Home , Artificial cell, Chemoton, Organism, Protocell

Synthetic : from to computation

Ricard V. Sol´e,1,2 Andreea Munteanu,1 Carlos Rodriguez-Caso,1 and Javier Mac´ıa1 1ICREA-Complex Lab, Universitat Pompeu Fabra (GRIB), Dr Aiguader 88, 08003 Barcelona, Spain 2Santa Fe Institute, 1399 Hyde Park Road, Santa Fe NM 87501, USA

Cells are the building blocks of biological complexity. They are complex systems sustained by the coordinated cooperative dynamics of several biochemical networks. Their replication, and computational features emerge as a consequence of appropriate molecular that somehow define what is. As the last decades have brought the transition from the description- driven biology to the synthesis-driven biology, one great challenge shared by both the fields of bioengineering and origin-of-life is to find the appropriate conditions under which living cellular can effectively emerge and persist. Here we review current knowledge (both theoretical and experimental) on possible scenarios of artificial design and their future challenges.

Keywords: cells, , , , ,

I. INTRODUCTION mer in the context of the latest developments and major objectives of the latter. Subsequently, we shall move from Cellular life cannot be described in terms of only general to particular, from this overall synthetic perspec- DNA (or any other information-carrying ) nor tive to the specific requirements of a minimal protocell. as metabolism or as compartment (cell membrane) alone. We shall review advances in the modelling approaches Cellular life emerges from the coupling among these three and also comment briefly on the experimental techniques components. A container is a prerequisite of biological that have drawn us closer than ever to achieving the goal organisation in order to at least confine reactions in a of SPB. limited , where interactions are more likely to occur (Deamer et al., 2002). It also provides a spatial location able to effectively facilitate division of labour among re- II. SYNTHETIC BIOLOGY actions. Moreover, in modern cells, the membrane is an active cell component, channelling nutrients in and waste As a direct consequence of late advances in molecular products out of the cell by means of specialised transport biology techniques and following the direction of the in- catalysts (Pohorille et al., 2005). A metabolism (Smith & creasing simulation-experiment , the discovery- Morowitz, 2004) provides the source of non-equilibrium driven biology of the last century is transformed and a mean of storage required in order to build into a hypothesis-driven biology. This approach made and maintain cellular components. It is also required to possible the control and design of new cellular func- allow cell growth to occur and eventually force splitting tions and genetic circuits (Sprinzak & Elowitz, 2005) and into two different (but similar) copies. marked the beginning of a new well-defined research field: If cells must adapt to a changing environment, informa- synthetic biology (Benner & Sismour, 2005). It reflects tion carriers will be also needed, as well as their coupling the idea that the best way to test the accuracy of the cur- with metabolic reactions. The fundamental problem, of rent biological knowledge is to modify or construct a dif- course, is how to obtain the coupling in such a way that ferent version of a biological and compare its be- self-reproduction of identical structures is achieved and haviour with theoretical expectations. As an inheritance self-maintained. Even though the scientific from (Ideker et al., 2001) and compu- has reach a consensus on the requirements and proper- tational (Slepchenko et al., 2002), synthetic ties of a minimal living system (Deamer, 2005; Pohorille biology uses a wide variety of mathematical descriptions & New, 2001), the materialisation of this vision into a including graph theory, Boolean logic, ordinary, partial concrete laboratory prototype is still incomplete. and stochastic differential equations. It is the solid feed- In this review we explore some of the methods, theo- back between an accurate and realistic modelling of the ries and perspectives related to the goal of constructing subject under investigation and the precise reproduction simple artificial cellular systems. The research program of the genetic or chemical design in the laboratory that to which this special number is dedicated and which we has recently granted synthetic biology a high-profile at- name synthetic protocell biology (SPB) aims at the con- tention in the scientific community (Andrianantoandro struction of a chemical life-like ensemble in the form of an et al., 2006; McDaniel & Weiss, 2005). artificial cell system able to self-maintain, self-reproduce There is still no clear distinction in the scientific com- and potentially evolve. The article is organised as follows: munity between synthetic biology and somewhat older as SPB can be considered as a field intersecting synthetic research fields, such as systems biology, biological or biology, which is a general framework encompassing sys- , recombinant DNA techniques tems biology and bioengineering, we shall place the for- (Brent, 2004). However, one might argue that the main 2 distinctive feature of synthetic biology seems to regard the emphasis on design and testing via simulation of new living biochemical systems endowed with complex be- haviour, followed by their experimental implementation. From this point of view, both synthetic biology and SPB follow the same course of action. Two main research branches can be delimited within the field of synthetic biology. A first one concerns the SPB: the assembly of synthetic units into chemical sys- tems endowed with biological properties, that is repro- duction, inheritance and (Pohorille & Deamer, 2002). The second branch aims at assembling biologi- cal units extracted from living systems and obtaining a modified or even an improved version of a (an existing) . This second branch confers a clear en- FIG. 1 The two main approaches to protocell building: top- gineering feature to synthetic biology by dealing with the down and bottom-up. Three potential classes of synthetic creation and rewriting of genetic circuits using building can result: self-maintained (non-replicating), repli- blocks (Endy, 2005). cating but not evolving and fully evolvable protocells (see One of the main goals pursued by both research text). branches consists in constructing, modifying and using biological mechanisms into performing desired functions, in other words, in obtaining a programmable plug-in ge- are involved in cell-cell communication while oth- netic device or biological entity (Kobayashi et al., 2004). ers have been shown to be non-essential to cell func- Either by a rational complete design (Weiss et al., 2003) tioning, it was early suggested that it would be possi- or by directed evolution techniques (using combinatorial ble to reduce complexity to a minimal set of synthesis: Yokobayashi et al. 2002), programming artifi- genes able to sustain (under given external conditions) cially designed living protocells appears as the world-wide cell life and reproduction. In this context, using the par- synthetic biology objective. In addition to programmable asitic bacterium as a case study, features, SPB also aims at integrating a property unique Mushegian & Koonin (1996) showed that approximately to living : evolvability. Even though there still is 256 genes seem to be needed to build a minimal a long way to go before achieving this objective, there is set that is necessary and sufficient to sustain the exis- active ongoing research on the evolutionary potential of tence of a modern- cell. No matter how small, cell protocellular and prebiotic structures (Sz´athmary et al., must contain all the information necessary for 2005; Yokobayashi et al., 2003). Moreover, the concept of the cells to perform essential (housekeeping) functions computation is intimately connected to both evolvability allowing them to maintain metabolic , repro- and programmability features and latest studies point to duce and evolve, the three main properties of living cells. it as a fundamental characteristic of biological systems Moya and co-workers (Gil et al., 2004) have also studied (Fern´andez & Sol´e, 2003). We shall discuss momentarily this problem using both genome comparison and compu- these concepts and the relationship between them in the tational modelling approaches, further reducing the list framework of SPB. of essential genes to only 206 (see also Gabaldon et al., 2006). In this paper we restrict our attention to the bottom-up III. BUILDING PROTOCELLS approach. Contrary to the top-down approach, it starts from scratch: a life-like entity is build by self-assembling The major works dedicated to SPB are organised in of molecular components (Rasmussen et al., 2003). These Table I. Even though SPB is a relatively young research can be of biological or instead completely ad hoc field, it is related to as well as a continuation of several chemical components. In both cases, a compartment is research fields and thus its frontiers are not clearly es- required, formed of some type of amphiphilic tablished. For this reason, the task of choosing among characterised by a natural tendency to make aggregates the most significant and relevant results obtained so far to which further cell components can join and couple. is extremely difficult. As a general rule, we chose to cite Plymers that mimic amphiphilicity can also assem- mainly review papers that incorporate direct citations of ble into vesicles, offering a much wider spectrum of prop- particular and detailed works. erties (Discher & Eisenberg, 2002). Two fundamental approaches have been considered to- To detail even further, Luisi et al. (2006) suggest also wards the synthesis of a protocell (fig. 1). The first the term reconstruction as the intermediate between the class, so called top-down approach (Luisi et al., 2002) bottom-up and the top-down approaches. This term involves the creation of a minimal cell by means of re- refers to the encapsulation of nucleic acids and ducing the genome of modern cells. Since numerous in defining thus a “semi-artificial cell” (Po- 3 horille & Deamer, 2002) whose self-replication is again sought in the experimental (Oberholzer et al., 1995) and theoretical works (Szostak et al., 2001). As indicated in fig. 1, the final outcome of both ap- proaches does not need to be a self-replicating, evolv- ing cell. The artificial cell (Acell) can replicate but not evolve, or it might even be unable to replicate itself: such simple self-maintained Acell would be able to persist un- der given conditions, exchanging energy and matter from its surroundings without growing. Although this seems a fairly limited situation, it is actually an interesting one: one could design or lead into self-assembling a protocell able to perform some given functions or computations in predefined ways (Pohorille & Deamer, 2002). These two major concepts, self-assembling and self-replication, FIG. 2 The logic of the self-reproducing machine: von Neu- pervade the field of SPB and origin-of-life and we shall mann’s self-replicating automaton components, closely re- discuss them below in more detail. lated to those found in living cells. See text for more details.

A. A self-replicating machine would be required. However, smaller versions of the sys- Since building a protocell using a bottom-up approach tem have been obtained even at the hardware level (Re- is not limited to biological, evolved ingredients, we should strepo et al., 2000). ask ourselves if very different combinations of the previ- One great implication of von Neumann’s solution was ous three ingredients (or none of them) might lead to the need for a copy mechanism for the instruction set. If life-like entities. To answer this question requires formu- no such mechanism were present, an infinite recurrence lating the problem under a theoretical perspective. Such problem would arise: the instructions would have to con- view was taken by the Hungarian mathematician John tain a replica of themselves which would be passed to the von Neumann in the 1940’s (von Neumann, 1966). In his new machine. Since he investigated the required logic of seminal work, von Neumann considered the logical condi- replication, he was not interested, nor did he have the tions under which an abstract - but embodied - automa- necessary tools, in building a working machine at the ton would be able to reproduce itself. The approach was bio-chemical or genetic level. Remember that at his fully computational: the reproducing system was viewed DNA had not yet been discovered as nature’s genetic as a machine equipped with building blocks and instruc- material. The fact that von Neumann’s approach was so tions. The solution found was innovative and visionary. close to current cell organisation is revealing. It suggests A basic scheme is shown in fig. 2. Here the basic building that universal principles might pervade the ways infor- blocks are indicated, including: mation, metabolism and container need to be coupled. We can identify the components of the automaton with 1. The constructor (A), able to build a physical, new those of living cells as follows: (a) the instruction set system by using the available raw material in the is the DNA molecule; (b) the duplicator is provided by surroundings. the DNA polymerase and other components of the cell’s 2. The blueprint or instructions containing informa- replication machinery; (c) the constructor corresponds to tion on what has to be performed by the construc- the RNA polymerase and the machinery (al- tor. lowing to be formed) and (d) the controller is nothing but the regulation of and transla- 3. A duplicator (B) which takes the instructions and tion. duplicates them accurately. The road initiated by von Neumann has been followed 4. The controller (C) required to guarantee that the by many other researchers over the last decades of the whole process takes place in some well-defined se- 20th century. As advanced, new tools quence. and methods allowed to consider a different approach to the problem of self-replication: replacing machines by Although not explicitly said in the previous descrip- biological components. In this context, several impor- tion, the automaton envisioned by von Neumann had a tant advances were obtained within the context of self- physical embodiment: he specifically thought on how to replicating molecules, both conceptually (Szathm´ary & build a physical system able to perform the whole repli- Maynard-Smith, 1997; von Kiedrowski, 1993) and experi- cation cycle. In other words, von Neumann’s system was mentally (see Paul & Joyce, 2004, and references therein). a machine. He designed a system which should have 29 These include different small-sized molecules able to dis- states and estimated that on the order of 105 elements play a closed replication cycle. 4

G G

ξ ξ D

R

D

FIG. 3 A schematic self-reproduction cycle of a protocell, requiring growth (G) and replication (R) phases in order to be completed. When dealing with a nano-scale scenario, both internal and external noisy fluctuations (ξ) are expected to affect the reliability of the whole process.

The machine’s self-reproduction envisioned by von Neumann has an equivalent picture within the context of protocell replication. The question here is: what are the H = H [S]dS (1) b I b conditions allowing a simple artificial protocell to reach S reliable reproduction? von Neumann’s picture includes In its simplest form and considering low temperatures (i. two key components of a complex able e. thermal fluctuations are ignored) we have to process information: hardware and software. In mod- ern cells, software is carried by DNA, whereas proteins play the role of cellular hardware. But in order to achieve κ[S] 2 Hb = (C(S) − C0(S)) dS (2) reproduction, no software is required (Dyson, 1999): if IS 2 the appropriate mechanisms of molecular assembly are in κ S C S − C0 S place and the system is able to spontaneously grow out where [ ] is the bending modulus and ( ) ( ) S from equilibrium, replication can occur without the de- is the mean curvature of the vesicle surface at . The signed concurrence of all the von Neumann’s components. result of the minimisation of such energy , i.e. In this context, the physical and chemical properties of the solutions of molecules are able to define a replicating entity without using the machine-like picture of sequential operations. δHb = 0 (3) In this self-organised picture, the basic cell cycle includes two steps: growth and division, summarised in fig. 3. allows to explain a number of basic vesicle shapes, from Both steps need the presence of non-equilibrium condi- red blood cells (Du et al., 2006; J¨ulicher, 1996; Piotto & tions unless some externally driven mechanism (such as Mavelli, 2004; Seifert et al., 1991; Waugh, 1996; Zhong- shear forces) is present. And they are affected by both can & Helfrich, 1987) to potential conditions for self- internal and external fluctuations, here indicated as noise reproduction (Boˇziˇc& Svetina, 2004; Svetina & Zekˇs,ˇ sources (ξ). Basic physical and chemical constraints are 1989). In a more general context, it can be easily shown the main players in this process, connected with the sta- that energy configurations forbid spontaneous splitting bility of membrane shape. To a large extent, it is the of a spherical vesicle to occur under the absence of other active breaking of such stability towards non-spherical energy sources (Sol´eet al., 2006). Below we will explore vesicle shapes what predates the problem of protocell re- some of the potential scenarios able to allow cell divi- production. This has been explored by a number of au- sion to take place. Evolution of cell division and other thors (Boˇziˇc& Svetina, 2004; Du et al., 2006; Jung et al., membrane remodelling mechanisms has led to sophisti- 2001; Seifert et al., 1991; Svetina & Zekˇs,ˇ 1989) and is cated mechanisms of organization and self-assembly that formulated in terms of the bending energy function Hb make them more and more independent of external fluc- associated to the closed vesicle. If we indicate as Hb[S] tuations (Shapiro & Benenson, 2005). However, much the free energy density at a given point S on the surface simpler protocells face a rather different situation, where S of the , the total bending energy will be the presence of noise can be an inevitable component to the integral over the cell’s surface S: be taken into account, particularly when dealing with nanoscale systems (Fellermann and Sol´e, 2006). 5

In von Neumann’s picture, self-reproduction was based on a deterministic sequence of events where the software and hardware of the machine interacted in a predictable way. When dealing with wet machines, we must take into account the spontaneous contribution of self-assembly processes and the constraints derived from them. We can also move away from the machine carrying instruc- tions to be copied. Here such information is embedded in the container and its interactions with metabolism and information, but the latter can also be removed from the system. Nevertheless, ongoing experimental efforts have explicitly considered von Neumann’s approach by using a small set of genes to be enclosed within a vesicle and truly acting as a software system able to sequentially con- trol a chain of events eventually leading to cell replication (Noireaux et al., 2005). FIG. 4 Some used in experimental ap- proaches to the synthesis of protocells based on vesicles as containers. In (a-b) two views of the α-hemolysin pore are shown. It has been used as a membrane channel to cre- B. Self-assemblying vs design ate selective permeability conditions in vesicles. Figure (c) shows the ATP synthase , spontaneously in- Most of these potentially feasible levels of protocell corporated to membranes and able to pump protons complexity are linked to vesicle containers. Thus, special inside them, thus allowing a simple metabolism to be main- attention has been dedicated to exploring the behaviour tained. In (d) the T7 RNA polymerase is shown, of these self-organised structures. Three decades ago, a with a small piece of the DNA chain also indicated. first crucial step towards the bottom-up synthesis of life consisted in the experiments on self-assembly phenom- ena leading to microscopic gel structures: Oparin’s coac- experimental designs. Some of them are shown in fig. 4. ervates (Oparin & Gladilin, 1980) and Fox’s protenoid These include transmembrane proteins able to facilitate microspheres. Early continuations of these scenarios of the active flow of precursors, special enzymes and poly- self-assembling prebiotic containers have focused on sur- merases taken from and and other well- factant assemblies into and vesicles. By now it is characterised molecules. These are of course only a few clear that the lipid vesicles are the meeting point of the items from a potentially huge of possible molec- top-down and bottom-up approaches, as the idea of cel- ular structures, and the ongoing progress of synthetic lular life evolving from within a compartment is univer- biology, with interacting sets of molecules defining oscil- sally accepted (Luisi et al., 1999). More precisely, experi- lators or switches, will help designing protocells able to mental works (Monnard & Deamer, 2002) complemented perform complex functional tasks. by computer simulations of self-assembly of amphiphiles (Fellermann & Sol´e, 2006) made the object of study in the bottom-up approach, while the top-down and reconstruc- C. Building scenarios tion studies focused on the more biological-like mem- brane structure called liposomes (Oberholzer & Luisi, As previously mentioned, the end point of SPB is the 2002). building of artificial cells able to behave in a life-like man- Liposomes are lipid vesicles (bilayers) typically pre- ner. This includes self-reproduction, self-maintenance pared from , the components of today’s and evolvability. But different intermediate stages can cells membrane. Besides being improbable if not impos- be also relevant, even if they do not incorporate all the sible that such molecules existed in prebiotic environ- previous ingredients. Before we present the different ap- ment, they confer a low permeability to the constituting proaches taken, a tentative list of artificial protocell types membrane and thus a high resistance to the uptake of can be defined as follows: nutrients (Deamer et al., 2002). Even in the absence of the evolved transport mechanisms of today’s living cells, 1. Self-maintaining protocells: in this type of syn- there are solutions to improve liposomes’ permeability thetic protocell, neither growth nor replication (Monnard & Deamer, 2001). From the point of view of would take place (e.g. Zepik et al. 2001). How- both origin of life and life synthesis, the use of fatty acids ever, active transfer of energy and matter can occur (surfactants) in membrane structures instead of phospho- by means of appropriate energy-transducing mech- allows a higher permeability to ionic solutes and anisms (such as transmembrane proteins pumping thus seams more plausible and efficient. protons) – Table I(5). Although this cell is unable In order to achieve the desired behaviour, researchers to replicate itself, it might have desirable proper- have also used a number of basic building blocks in most ties such as acting as a nanomachine able to process 6

TABLE I Protocell studies in the literature. The review works appear in italics. Approach Container Bottom-Up (1) Lipid world Segr´eet al. (2001) (2) Self-catalytic lipid aggregate formation. Theory & Simula- Mavelli & Luisi (1996) tions (3) Self-catalytic lipid aggregate formation. Experiments Bachman et al. (1992); Berclaz et al. (2001a,b); Chen & Szostak (2004); Hanczyc & Szostak (2004); Takakura & Sugawara (2004); Takakura et al. (2003) (4) Self-catalytic lipid aggregate formation. Simulations Chen & Kim (2004); Lyubartsev (2005); Nils- son et al. (2003); Noguchi & Takasu (2002) (5) Increasing bilayer permeability Monnard & Deamer (2001) . channels: Noireaux & Libchaber (2004); Pohorille et al. (2005) . microtubulation: Roux et al. (2002) . alternating electric field: Fischer et al. (2002) (6) Thermodynamics of self-assembling of lipid aggregates Mayer et al. (1997) (7) Fusion of liposomes/vesicles Pantazatos & MacDonald (1999) (8) Photoinduced vesicle formation Veronese et al. (1998) Biochemical reactions in container. Reconstruction (9) Conceptual framework of macromolecules encapsulation Szostak et al. (2001) (10) Protein expression in liposomes Oberholzer et al. (1999) (11) Increased efficiency of (transcription of T7 Nomura et al. (2003); Tsumoto et al. (2001) DNA) in liposomes (12) Two-stage genetic network encapsulated in liposomes Ishikawa et al. (2004) (13) Enzyme-driven vesicle replication Hanczyc et al. (2003); Szostak et al. (2001); Walde et al. (1994) (14) Coupled RNA-template and vesicle replication. Experi- Chen et al. (2004); Oberholzer et al. (1995) ments (15) Homeostatic behaviour Zepik et al. (2001) (16) Encapsulation of E-coli cell-free expression system in lipo- Noireaux & Libchaber (2004) somes Bottom-Up (17) Coordinated shell-core growth. Simulations Munteanu et al. (2006); Serra et al. (2006 (In press) (18) Photochemically driven shell-core growth & replication Rasmussen et al. (2003) Information (I) / Metabolism (M) / Container (C) (19) MC System Cronhjort & Bloomberg (1997); G´anti (2003) (20) CI System Szostak et al. (2001), Fontanari et al. (2006) (21) MCI System. Simulations Fernando & Di Paolo (2004); Munteanu et al. (2006); Zintzaras et al. (2002) Evolutionary potential (22) General implications of autocatalytic subsystems Kaneko (2005) (23) Minimal protocell as unit of evolution Sz´athmary et al. (2005) (24) Evolving replicators in limited dispersion Szab´oet al. (2002) Top-Down Minimal cell (25) Generalised kinetic model of E Coli metabolic mapping Browning & Shuler (2001) (26) Conceptual framework of minimal gene set Gil et al. (2004); Koonin (2000); Luisi et al. (2006, 2002) (27) Experiments on minimal genome Hutchinson et al. (1999); Mushegian & Koonin (1996) (28) Comparative Koonin (2003) Computational potential (29) Protein molecules as computational elements Bray (1995) (30) Design of genetic circuits Fran¸cis & Hakim (2004); Savageau (2001) (31) Programmable functions You et al. (2004) 7

R R R R 2 R Lµ R 1 Lµ R A1 R g 1 Rµ 1 Rµ W W R R A1 A 1 1 1 R R 2 S2 L R Lµ g Lµ S 1 R 1 A 4 A2 g 2 L R2 L 2g 0 Lµ Lµ L A3 W g R R R1 2 2 Lµ R R 2 2W W R A1 1 R R2 L Lµ L 2 W W R1

R R R R R R R R R R* R * L R R ATP Rz2 L ATP R + R H R RNA 2 + R R H R R * ADP POL r r cRz2 R R h ν R R POL POL POL R R RNA 1 R* R CPOL R R Rz2 ATP bR POL + DNA R r + H R r R H ADP RNA R RNA 2 R L R R R R RNA 1 R Rz2 R R R R DNA R R R R R

FIG. 5 Examples of protocell models involving a well-defined membrane separating the external environment from the inner volume, where reactions take place. These include: (a) simple systems with a single replication cycle, (b) Turing protocells, exploiting spatial instabilities as a source of membrane change, (c) the , (d) synthetic cells involving , (e) minimal protocells including an internal compartment performing energy and (f) minimal cells with all the components found in complex cells.

matter or information under given external condi- The simplest scenario does not include information- tions. A biosensor or a system can carrying molecules and thus does not consider evo- be potentially implemented at this level. lution. However, in spite of the limitations im- posed by evolution-free systems, it might actually 2. Growing vesicles: due to active transformations be important in a number of applications (such as of external precursors, growth can take place over biomedicine) where evolvability is actually some- some transient time. These systems eventually thing to be avoided. stop growing (once an equilibrium between inter- nal metabolism and matter exchanges is reached), but can be helpful in exploring through both exper- 4. Replicating protocells: “reconstruction” approach is iments and simulations the mechanisms that allow probably one of the most active directions in SPB, self-aggregates and artificially designed vesicles to where macromolecules are encapsulated in vesicles grow – (Chen & Szostak, 2004) Table I(1-8). This or liposomes and catalyse metabolic functions nec- system can also be a first step towards cell division essary in the life cycle of the protocell. One such by using extrusion mechanisms through membrane example is Szostak’s theoretical minimal RNA cell, pores – Table I(13). not yet implemented in laboratory in the exact composition as prescribed by Szostak et al. (2001). 3. Replicating vesicles: if vesicles or micelles are However, different versions of the RNA cell have driven out from equilibrium and keep growing, been created in the laboratory (Bartel & Unrau, they can eventually reach unstable configurations 1999; Chen et al., 2004). In the long list of exper- favouring membrane , deformation and imental works, an important place is held by the eventually division (Hanczyc & Szostak, 2004). works of Walde et al. (1994a) and Oberholzer et al. 8

(1995) showing “core-shell” replication in designed Additionally, surrounding lipid molecules L become protocells. This coordinated replication of both incorporated into the vesicle as membrane bounded container and metabolites (and/or genetic mate- molecules (here indicated as Lµ). Since the process rial) is necessary in order to avoid by dilution is necessarily linked to enzymatic activity, the split- after several generations of protocells. Different ting is a single event, not able to be repeated. from the reconstruction approach, the Los Alamos Bug model (Rasmussen et al., 2003) and versions 2. Turing-like protocells: these model proto- of it, based on the self-assembling approach, have cells introduce a very simple coupling between been proved to fulfil this condition, an emergent a reaction-diffusion system defining a metabolism property of the catalytic coupling of protocell’s sub- and membrane dynamics (fig. 5b). In this scenario systems – Table I(17). Opposed to these catalysis- there is no need for spatially localised enzymes. based protocell models, a stoichiometric model of Instead, externally provided precursors R1, R2 are a protocell, the chemoton model (G´anti, 2003) ac- supplied, entering the membrane and being trans- complishes the coordinated growth of all its compo- formed into new molecules through a set of sim- nents by means of a precise imposed stoichiometry ple reactions. The reactions inside and outside the in the transformation nutrients-metabolites-waste. cell are represented by a set of n reaction-diffusion (RD) equations (Murray, 1989) namely: 5. Evolving protocells: if the macromolecular - isation of the SPB includes an information car- rier coupled to metabolic and container dynam- dCi 2 =Φ (C1, ..., C )+ D ∇ C , (4) ics, the whole assembly can experience evolution- dt i n i i ary changes and Darwinian selection (Sz´athmary et al., 2005). For example, in the stoichiometricaly- with i = 1, ..., n, the index associated to the i-th coupled chemoton model (G´anti, 2003), the infor- morphogen having a local concentration Ci. Each mation is carried by of a given length, term Φi describes how the i-th molecular and thus changes in the number of constituting reacts to the other molecules. The last term in monomers can induce changes in the efficiency of the right-hand is the diffusion term accounting for protocell dynamics and thus a genotype- the spontaneous, random movement of molecules mapping. through space. However, the formalism needs to be extended by incorporating a changing bound- ary which now acts as a permeable membrane, also IV. PROTOCELL MODELS coupled to the reactions described by Φi. These re- actions will define the protocell metabolism. Since In the context of the scenarios previously mentioned, osmotic pressures are associated to differences in several models of synthetic protocells can be envisioned. molecular concentrations, active mechanisms gen- In this section we enumerate some possible examples of erating spatial heterogeneity are expected to cre- systems that allow cell self-replication by using different ate changing pressure fields. These instabilities potential mechanisms. Some of these systems have been can break the symmetry along the explored from the mathematical point of view. membrane, and after division the reactions defin- ing the metabolism must be able to trigger a new 1. Enzyme-driven, information-free protocells: growth-division cycle (Mac´ıa & Sol´e, 2006b). based on an early suggestion by the Russian biomathematician Nicolas Rashevsky, it has been 3. Chemoton-like protocells: the chemoton intro- shown that the stability of a spherical closed mem- duced by G´anti (1975) consists in three functionally brane can be lost by providing an appropriate set dependent autocatalytic subsystems: the metabolic of metabolic reactions (Rashevsky, 1960). Specifi- chemical network, the template polymerisation cally, Rashevsky conjectured that a vesicle having and the membrane subsystem enclosing them all enzymes allowing metabolic reactions to occur in- (fig. 5c). The self-reproducing metabolic network side the compartment could experience a destabil- transforms the external nutrients into chemoton’s isation eventually leading to cell division. It has internal material necessary for template replication been recently shown that this is the case for a very and membrane growth. The correct functioning of simple model involving, for simplicity, two enzyme the chemoton lies in the precise stoichiometric cou- molecules, placed at the two opposite poles of the pling of the three subunits, more precisely the co- cell (fig. 5a). If these enzymes catalyse a reaction ordination between the accumulation of molecules transforming a given precursor (R in figure 5a) into and the surface increase in order to achieve an equi- a new molecule G, then close to the location of each librium of the osmotic pressure relative to the envi- enzyme, the concentration of G would increase and ronment. The model imposes a closed stoichiomet- eventually trigger a heterogeneous pressure distri- ric coupling of the autocatalytic cycles such that bution along the membrane (Mac´ıa & Sol´e, 2006a). the number of membrane molecules necessary for 9

surface doubling is equal to the number of poly- to the bottom-up approaches, there is still a lot merisation iterations needed for a complete repli- of experimental work needed in order to establish cation of all double-stranded template molecules. tenable and incontestable rules of thumb for the be- As shown by recent works (Table I – 17), the sto- haviour of genetic modules within the genetic regu- ichiometric coupling is not necessary for fulfilling latory networks. Latest advances in synthetic biol- coordinated growth as the catalytic coupling of the ogy have revealed opposing facts: that there is an subsystems can ensure their coordinated growth, enormous potential for designing and building pro- and thus the doubling of their components prior to grammable genetic devices, on one hand, and that division, which is the condition for a viable replica- surprises are to be expected when passing from sim- tion cycle. ple circuits to devising multimodular or hierarchical genetic networks as a consequence of emergent new 4. Ribo-cells: (fig. 5d) Szostak et al. (2001) and behaviours of numerous interacting agents, on the Bartel & Unrau (1999) have suggested possible other hand. Thus, the step from simple genetic cir- RNA protocells under the form of minimal ribo- cuits to a new artificial cellular entity has to deal : one encapsulated would syn- with both the unlimited behavioural diversity and thesise the vesicle membrane component, and a sec- the precision in reaching the programmed target ond ribozyme would replicate itself and the first intended for the artificial cell. one. The components necessary for the RNA imple- mentation as seen in these works are not yet avail- Most of these basic cell models can be used to im- able, and there are clues suggesting that a DNA plement computational tasks. Here computation means cell might appear easier to implement experimen- some sort of predictable response to external signals. In tally than “simpler” RNA cell (Luisi et al., 2002). figure 6 we show two simple examples of explicit designs However, a different RNA cell has been proposed of two basic logic gates (the NOT and NOR gates, re- and implemented in the laboratory by Chen et al. spectively) from the first model described in this section. (2004) showing that RNA replicating within vesi- Here the external signals correspond to some type of in- cles could increase membrane growth rate by creat- hibitors of enzyme activity. As a measure of the output, ing internal osmotic pressure. An even bigger step we use cell division: the output D will be one if division towards the prototype of the minimal protocell was takes place and zero otherwise. In other possible scenar- accomplished by Ishikawa et al. (2004) through the ios, the output can correspond to some type of produced laboratory implementation of a two-level cascading molecule resulting from cell reactions. protein expression in liposomes. The simple struc- The NOT gate can be obtained by using an enzyme ture of ribocells allows them to be fully described having an single inhibitor I: under the presence of I no by means of whole-protocell simulation models (see division occurs, whereas if absent the vesicle experiences for example Flamm et al. 2006). reproduction. A simple generalisation of this using two enzyme inhibitors (here indicated as I1 and I2) leads to 5. Transduction-driven protocells: Efficient pro- a NOR gate: unless both are absent, no reproduction tocellular systems incorporating both DNA and can occur. Although these are rather trivial examples, RNA (fig. 5e) could be obtained by incorporating they illustrate possible ways of designing simple types of appropriate energy transduction systems (Pohorille computational cell structures. Once we incorporate as et al., 1996). One such subsystem appears indi- inputs the produced molecules or alternatively introduce cated within the protocell in fig. 5e. It consists of different types of cells responding to different signals, it two proteins, bacteriorhodopsin (BR) and the F0F1 is not difficult to generate (at least at this theoretical ATPase from the thermophile Bacillus PS3, embed- level) more complex systems able to describe switches ded in liposomes (Richard et al., 1995). BR is a or even memory structures. It is worth mentioning that light-driven proton pump. It generates the trans- very complex computational devices (including Turing membrane proton gradient required for the ATP- machines) are currently being developed experimentally making activity of the ATPase. It has been shown at the molecular level (Shapiro & Benenson, 2006). These to have high turnover and stability. This would be a molecular automata might benefit in the future from the minimal protocell incorporating compartments in- current advances in synthetic protocell biology. side its structure, thus making metabolism to be effectively associated with a special protocell sub- structure. V. DISCUSSION

6. Minimal cells: the concept of minimal cell The previous models and theoretical approaches to (fig. 5f) is the target of the works employing the protocellular systems define a range of complexity lev- top-down approach, as mentioned in the beginning els from non-evolving, non-replicating structures to fully of the previous section. Even though it appears as reproducing and evolving entities. So far none of these a more promising and straight-forward strategy in systems has been shown to exhibit autonomous reproduc- the search of a minimal protocell, when compared tion. Achieving such goal would represent a great leap 10

a R E G b E c G I NOT logic gate G G R R G G G R R G G GG G I D R G R G G R

R R ID

G L 0 1 G R R I G G 1 G R G I 0 R L G G G G G G E G G E

d e f NOR logic gate E E I 1 I 1 I 2 D I 2 R R I 1I 2 D R 0 0 1 R R R 0 1 0 R I R 1 1 0 0

I 2 R 1 1 0 E E

FIG. 6 Protocells could be used to perform simple types of computations. Here two very simple examples are given, for illustrative purposes only. Computation is linked to cell division (D): as the output to some given inputs, the cell can (D = 1) or cannot (D = 0) replicate itself. Here we use a protocell model (Mac´ıa & Sol´e, 2006a) where two enzyme molecules (E) are used as catalytic systems, transforming an external precursor R into a new molecule G. If the enzymes can be repressed by using given inhibitor molecules (I) then simple switches can be made. In (a-b) we show how to implement a NOT gate (c), whereas the second example (d-e) illustrates a more complex, two-input gate (the NOR gate). in biology: it would provide the logical basis for under- through membrane proteins. Their enormous versatility standing the nature and requirements of life at its sim- allow the potential exploration of a highly diverse uni- plest level. All the evolutionary advantages of cellular life verse of biomembrane designs. (from compartmentalisation to division of labour) would enhance the potential advantages of synthetic molecular The success of any of the previous model approaches systems. (and perhaps others not considered here) will provide the It is important to mention that possible scenarios of basis for a new field at the crossroads between biology vesicle change in nature are not restricted to whole cell and computation. Travelling from non-living to living changes associated to reproduction. A wide diversity of matter means to cross a twilight zone: some transition mechanisms of membrane formation and processing ex- where the preconditions for reliable cell replica- ist inside complex cells, representing a variety of possible tion (and thus life) exist. Although some steps need to forms of building vesicles. These include transport dy- be completed and some key processes are not yet under- namics using Golgi vesicles, endosomes, lysosomes and stood, we are likely to see the success of synthetic cellular clathrin-coated vesicles (Alberts et al., 2002). Inspira- life soon at work over the next decade. tion from such processes might help designing new types of protocellular systems. Some of these processes include the participation of the , a complex nanoma- Acknowledgments chine involved in reading RNA molecules and translating them into proteins. The computational power of ribo- somes, combined with designed protocellular systems ex- The authors would like to thank the members of the ploiting spontaneous pathways of vesicle dynamics might Complex Systems Lab for useful discussions. Special help expanding the current state of the art in this area. thanks to Jurgen F. Sebastian for useful discussions. This Additionally, vesicles obtained from non-biological poly- work has been supported by grants FIS2004-0542, IST- mers have been shown to allow interfacing with biologi- FET PACE project of the European Community founded cal structures (Discher & Eisenberg, 2002). These poly- under EU contract FP6-0022035 (Programmable Artifi- mersomes can mimic many biological processes, including cial Cell Evolution) and by the Santa Fe Institute. encapsulation of relevant molecules and transfer-loading 11

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