Origin of life from a maker's perspective -- focus on protocellular compartments in bottom-up synthetic biology

The origin of life is shrouded in mystery, with few surviving clues, obscured by evolutionary competition. Previous reviews have touched on the complementary approaches of top-down and bottom-up synthetic biology to augment our understanding of living systems. Here we point out the synergies between these fields, especially between bottom-up synthetic biology and origin of life research. We explore recent progress made in artificial cell compartmentation in line with the crowded cell, its metabolism, as well as cycles of growth and division, and how those efforts are starting to be combined. Though the complexity of current life is among its most striking characteristics, none of life's essential features require it, and they are unlikely to have emerged thus complex from the beginning. Rather than recovering the one true origin lost in time, current research converges towards reproducing the emergence of minimal life, by teasing out how complexity and evolution may arise from a set of essential components.

The question how life originated is difficult both because direct evidence is long gone and because modern definitions of life reveal it is a grayscale continuum phenomenon, not a binary yes/no test. One humbling yet motivating point of agreement is that life is a complex phenomenon that is not sufficiently understood and not yet possible to build in the lab -despite the Miller-Urey experiment 1,2 and the technological advancements in combinatorial chemistry, 3,4 fast-forwarding billions of years of stochastic conditions and interactions on a planetary scale. In an effort to strip down the skeleton of life, top-down synthetic biology approaches have modified living cells by, e.g. paring genomes down to a set of 473 genes capable of supporting replication, though the functions of a third of those is still unknown. 5 Bottom-up synthetic biology, in contrast, starts with few components, performing increasingly complex functions, with the aim of creating life. 6 Furthermore, it has the ability to take under its umbrella research from a number of scientific fields such as colloidal science, biochemistry, biophysics, genetic engineering, active matter, etc. and can be considered as a conceptual offspring of abiogenesis research. Thus, it has inherited many of the tools and aims of the latter, and the terms proto-, synthetic, artificial, and minimal cell are often used interchangeably. However, bottom-up synthetic biology is not burdened by the command of the evolutionary timeline -its molecular toolbox ranges from simplistic presumable predecessors to highly evolved biological machinery. It takes advantage of the newest developments in molecular biology and relies rather on controlled production, than on environmental cycles and chance. In addition, it is not limited to natural building blocks and architectures but augmented by the power of synthetic chemistry and subjected to modular reorganization at will, with the overarching aim of reproducing essential biological features with a minimal set of parts.
Similar two-sided approaches in the exploration of life's emergence and evolution are attempted all the way from defining the chemistry of proto-life, to the higher functions above mostly considered the domain of biology, with a palpable tension. On one hand there is a desire to "recognize life when we see it" often implying not only DNA but most of its other modern properties, as if in the Greek myth seeing Aphrodite (Venus) emerging fully formed from the ocean foam 7 . On the other, it is a self-evident postulate that the intricate constituents and complexity of modern biology did not emerge at once in that prebiotic time.
Therefore, it has been necessary to separate and conceptualize the component functions by which we might recognize life more abstractly, including not only the above movement, metabolism, communication, reproduction, and evolution. Paring down the origin of more advanced cell properties, such as compartmentation, current metabolic cycles, or the present DNA-RNA-protein machinery will bring additional fundamental insights but may also trigger new applications. For example, minimal chemical systems have been proposed to randomly generate predecessors and plausible molecular pathways to the molecules of life 4 . But such systems also have the highly sought application of designing the synthesis for any molecule. Equally simple "biological" ensembles are put together from few components to mimic ever larger collections of features of life. In both chemical and biological systems (we note the increasingly arbitrary discrimination) scientists aim to reproduce central life processes, while hunting for emerging phenomena, self-assembly, reaction networks with autocatalytic properties, etc.
Furthermore, life is energy-demanding and from a thermodynamic point of view requires constant external drive to maintain its out-of-equilibrium state. However, even minimal chemical systems may exhibit complex energetic transformations, leading to apparently living phenomena. For example, twocomponent swimmers in water recently demonstrated a novel elastohydrodynamic propulsion mechanism and showcased that even such simple systems can harness energy from temperature fluctuations and store it to recharge 8 . Thus more and more complex processes are moving out of the exclusive domain of biology.
In this chapter, we start with a comparison of the two, in our opinion complementary, syntetic biology approaches, followed by discussion of selected advances in the bottom-up approach. We show how bottom-up synthetic biology combines physical, chemical and biological tools to yield ever more complex artificial structures in a rationally directed and accelerating evolution process, driven by human ingenuity, which may turn out to be a more instructive, definitive, and functional approach than historical speculation. Thereby, our aim is not a comprehensive literature overview but to present a few major developments, revolving around compartmentation, which exemplify the reconstitution and understanding of key living features via minimal synthetic systems made of both natural and man-made constituents. We also attempt to address the limitations and the missing parts in the present blueprint for assembly of an artificial cell as the pieces of a yet undefined puzzle.
The question of origin of life looms large in the last decades and there is optimism among researchers that the problem of synthesizing life in the lab can be solved in the next few decades. Several scientific fields are trying to approach the question from different angles, and synthetic biology has taken a relatively new role in this endeavor. The latter research field has long been dominated by a more top-down approach, modifying existing life and thus developing biotechnological solutions of great practical importance. On the other side, bottom-up synthetic biology -a recently coined and still not firmly established termshares many of the aims and problems of the origin-of-life research even though in its current iterations it may seem removed from its conceptual ancestor. However, we believe that the ability to build complexity from few components is key to answering "what is life?", and thus to creating life via abiogenesis. All of synthetic biology struggles with the overwhelming complexity of biology, which in turn often seems to doom both approaches' efforts to dead ends. Consequently, for better understanding and control they both resort to the fundamental scientific method of reductionism, too. We illustrate the central features of both concepts in Fig. 1, and compare them in the chapter. The top-down approach always starts with living biological cells and reduces their complexity step by step, while still monitoring principle viability as the decisive feature. It dominates the current perception of the term "synthetic biology" in the biosciences, and also in the society, partly because it has made practical advances in sophisticated genetic engineering methods 9 . The stripping down of genes in that approach to expose the functional skeleton of life, however, has been daunting and not ready yet to serve as a simple chassis on which to install new features. For example, the celebrated genome reduction of Mycoplasma mycoides has yielded a "minimal cell" with 473 genes, acknowledging that hundreds (one third) of these are with still unknown function 5 . Thereby, the top-down requirements for success have been somehow more generous since they are defined not by understanding but by apparent biotechnological figures of merit, e.g., the production of a given protein, and not by mechanistic blueprints from molecular and structural biology that serve as incomplete instructions for building a cell. On the contrary, bottom-up synthetic biology usually addresses initially non-living systems with up to 20 chemicals in a bilayer membrane or other physical confinement, where most of the component interactions are known. Remarkably, such dramatically simplified systems have recently been brought to successfully mimic several curiously complex features of living systems. These include motility 8 , metabolism 10 , communication 11 , reproduction 12 , and evolution 13 , as covered in more detail below. There are also combinations of both top-down and bottom-up approaches, which have intermediate complexity -for instance, ready-to-use protein expression machinery in form of a cell extract, encapsulated in minimal compartment. For the purpose of engineering features of life, the bottom-up approach within synthetic biology has an unlimited arsenal of building blocks at its disposal. Thus, next to primitive molecules from the speculative toolbox of abiogenesis, highly evolved or engineered protein machinery and even fully synthetic alternatives, products of modern chemistry, are being employed. The bottom-up method aims to pick up prokaryotic and eukaryotic functional units like LEGO pieces, borrows proteins across all kingdoms, tunes and blends, dissembles and reassembles them in minimal or novel ways. In parallel, synthetic chemistry and material science deliver additional means, deliberately or accidentally biomimetic, which may fuel bold speculations about other possible life forms 14,15 . Here, the creative aspect during the quest for the physicochemical principles of life is particularly emphasized, not only as a search for what happened long ago, but how we could use building blocks to start life de novo. In other words, studies of molecular assemblies with life-mimicking collective properties should not only help defining life and understanding how it appeared, but will also allow building a living cell, or possibly a tissue, organ or even organism from scratch. Furthermore, ever more complex biological phenomena are becoming addressable in the bottomup approach, enabled by the evolved molecular toolbox.
Unlike the abiogenesis research, which firmly serves a fundamental role, bottom-up synthetic biology is able to incorporate parallel efforts in colloidal and material science, which also have great application potential. In the long run, once we learn what life is, how to put it together and jump-start it, we may succeed in engineering the perfect platform organism -assembled and tuned to maximize yields and minimize the metabolic penalty for self-preservation, unchained from the muddle of evolutionary heritage. Both top-down and bottom-up approaches are driven in parallel towards this goal.
Metaphorically speaking, the top-down biologist pulls cards from a house tower until it falls, while the bottom-up artisan stacks cards until the tower is raised. To tackle this problem, both lines of research in synthetic biology employ a modular tactic, divide et impera, which is conceptually reminiscent of conventional mechanical and chemical engineering, hence the parallels to building a car or a chemical plant. In fact, we argue that the modularization of the traditionally fluid, interpenetrating and messy concept of life is one of the hallmarks of the umbrella term synthetic biology. The other defining feature is directly evident from the semantics of "synthetic" and exemplifies the desire to assemble and create at will, analogous to the established field of synthetic chemistry.
Reductionism is a valid and proven method, intrinsic to science, and not exclusive to any research field.
Bottom-up synthetic biology tries to reduce biological complexity by creating putative biological entities with certain key functions of life. It can be considered as an evolved form of traditional in vitro reconstitution experiments in biochemistry, enabled by continuous technological developments and accumulation of knowledge. We note that abiogenesis is not the only motivation for bottom-up synthetic biology and that such experiments build up the understanding of cells and biology in general. We note in the same way, that top-down synthetic biology has been characterized as nothing but a more powerful genetic engineering, continuously evolving since the first attempts of selection and cross breeding.
However, although there may be some truth in synthetic biology being partial rebranding of other fields, we believe that there is increasing momentum and awareness for an impending paradigm shift. Its connection with the famous Feynman quote "What I cannot create, I do not understand" will have multiple implications for life sciences and biotechnology. Thus, by creating and understanding new systems, synthetic biology will help reveal the underlying molecular and physical principles of life and will guide the elucidation and discrimination of conflicting abiogenesis hypotheses.
In Table 1, we present a brief summary comparison of the tradeoff made between the top-down and bottom-up approaches in terms of functionality and understanding. Though not alive, the featured bottom-up constructs have drastically fewer, well-characterized components, to which one can reduce the mechanism for the functionality in question and possibly understand the component actions. Below we present a concise kaleidoscopic overview of some recent efforts towards the creation of a cell, termed interchangeably proto-, minimal or synthetic, largely depending on the preference of the researchers. We focus on recent progress milestones in compartmentalization, reflecting our own and attempt to relate to other key abiogenesis questions like metabolism and cycles of growth and division.
Wefirmly refrain, however, from claims for comprehensiveness, let alone favoring metabolism-first or RNA-first hypotheses.

Unifying the plausible protocells in line with the crowded cell
Some form of compartmentation is indispensable for life, even if it should only serve to overcome the dilution of molecules. In fact, compartments do much more -they segregate reactions, enable gradients and bestow individuality, among many other functions. From the simplest protocells, compartments have evolved to today's highly compartmentalized and crowded cells. There are many parallel efforts in trying to decipher their physicochemical and biological origins, and whether membranes came first, or the liquidliquid phase separation of coacervates. Bottom-up synthetic biology (as a conceptual offspring of the origin-of-life research) has generally not been concerned about the evolutionary timeline and prebiotic plausibility of their building blocks 24 . But by constructing more and more functional models of life, it will show their relative importance for de novo origins of life.
Thus, vesicles are being formed from phospholipids, their presumable predecessors fatty acids, and combinations thereof, but also from synthetic molecules such as amphiphilic polymers 25 , showcasing the omnipotent molecular principles of self-assembly. These polymersomes, as well as their hybrid combinations with phospholipids, substantiate the chemically synthetic aspect of bottom-up biology, striving to achieve improved properties by deliberate design of building blocks 26,27 . However, they also mimic fundamental phenomena like membrane phase separation 28,29 , associated with the raft hypothesis as a means for two-dimensional compartmentation of membrane proteins, which has in turn numerous implications for the organization of life. Different vesicles are now routinely used to accommodate advanced biological processes like mimicking replication via PCR 30 or reverse-transcription PCR 31 and protein synthesis 32 .
Alternatively, it may be reasonable to assume that phase separation of an active "proto-cytosol", potentially stabilized by viscosity, has preceded the membrane as a universal boundary, or at least that both variants of compartmentation emerged simultaneously in a synergistic manner. This hypothesis is reinforced by the recent findings and emerging consensus on the ubiquitous role of liquid-liquid phase separation in biology 33 . In parallel to sequestering nanoparticles and biomolecules, and the enhancement of enzyme kinetics 34 , coacervates may be coated with fatty acids as a primitive form of a membrane 35 .
Such an architecture bridges the apparent dichotomy (largely determined by the preference of the researchers and not so much by conflicting hypotheses) with respect to possible prebiotic compartments, and has been realized with phospholipids and short polyamines 36 but also with fully synthetic polycations 37 .
An interesting but still underrepresented aspect is the deliberate control of compartment morphology, as both membraneless and membrane-bound compartments tend to relax to spherical shapes. Thus, the transient non-spherical shape of biomolecular condensates 38 has not been arrested yet, unlike the diatom-reminiscent morphology attainable in hydrocarbon emulsions 22 . Notably, though the exact mechanisms are still under debate and investigation, 39 the causes of shaped archaea may be due to physical phase transitions rather than biological programming 24 patterns and three-dimensional structures. 50 The process of artificial morphogenesis has been used to grow prescribed geometric shapes bottom-up -oil droplets transforming due to internal phase transitions -has been shown to apply to a variety of oils 51 , some used in biology, which may have been the basis of protobiological shapes. The smallest genome of a modern non-spherical bacterium so far is of the helical Drosophila symbiont Spiroplasma poulsonii sHy, with 1584 genes 21 , though mapping between genome sizes and shapes of organisms is an open problem that could shed some light on which shapes are more difficult than others.

Self-sustained cycles of growth and division
In addition to structures that help sustain it, life is composed of highly dynamic and far out-of-equilibrium processes, which both origin-of-life studies and bottom-up synthetic biology seek to reconstitute. These range from a transient single biochemical reaction 52 to complex interactions and feedback loops enabling oscillatory behavior 53 . Separately, the processes of growth, copying of genetic information, or division have recently been achieved in many contexts. However, the combined process of reproduction of growing entities together with heritable information has been elusive. It is notable that of 7 genes determined required for a normal cell division of the smallest cell so far, only two (ftsZ, sepF) have known function 16 . Thereby, continuous self-reproduction deserves a particular attention, as it is recognized as one of the cornerstones of life and constitutes the basis for autopoiesis, heredity and evolution and thus it is a fruitful area of current inquiry. We provide a short overview of milestones in this direction, as well as other recent perspectives of approaches, suggesting possible solutions of the problem 54,55 .
With respect to self-reproduction of protocellular compartments, consecutive cycles of growth and division have been demonstrated predominantly in membrane-bounded compartments. This is also the more challenging case when compared to coacervate growth, where existing frameworks from emulsions (e.g., Ostwald ripening) can be readily applied. The growth of fatty acid vesicles by simple uptake of the same is experimentally attainable thanks to the fast dynamics of these simpler amphiphiles 56 . In some cases, it is succeeded by division 57 , while for instance, the temporal coupling with RNA replication 58 would be a matter of synchronization. The realization of primitive strategies for growth of phospholipid vesicles, however, is not as straightforward 59 . In this regard, bottom-up synthetic biology may force the revisiting of mixed approaches, involving both single-and double-chain amphiphiles 60 . Even though the motivation behind such studies might have been different in the past, mixed phospholipid-fatty acid systems bear significant biological resemblance, related to the uptake of exogenous fatty acids and their use for membrane synthesis. In this regard, phospholipid vesicles were shown to grow upon addition of oleic acid, which was then converted to phospholipids by an in vitro assembled eight-enzyme cascade 61 . Similar cascades, encapsulated in liposomes, have been also encoded, translated and even self-regulated via genetic programming of cell-free systems 62 . Cytosol-confined transcription-translation machinery has been also used to grow peptide compartments 63 , where the somehow exotic membrane building blocks bear a remote analogy to viral capsid proteins. Polypeptide compartments have been also shown to fuse and used to accommodate DNA ligation 64 . Recursive reproduction of liposomes has been coupled with RNA replication via freeze-thaw cycling over ten iterations 65 . While this approach is not cell-centric but populational, in the sense that the progeny cannot be traced because the reproduction relies on random exchange of membrane and nucleic acids, it holds abiogenetic relevance with respect to the messy conditions at the onset of life. In parallel, membrane growth and division has been realized via synthetic precursors, at the borderline of biology. In one example, the membrane self-reproduction was coupled with DNA amplification 66 , where nucleic acids participated in the formation of the catalytic complex, while the combination with fusion also allowed for recursive cycles, referred to as ingestion, replication, maturity and division 67 . In another report, triazole derivatives were used as phospholipid-embedded autocatalysts that drove potentially inexhaustible membrane formation 68  other indirect (protective) advantages for the latter 82 . We note that although many of the currently investigated coacervates rely on nucleic acids, other "life-inert" phase-separating molecules 83 may be also sought as potential seeds for life "crystallization".
In an oversimplified analogy to modern cells 84 , once the protocell has metabolized and acquired sufficient material, it is ripe to split into two. Thus, next to reconstitution of membrane formation, one of the other current challenges in bottom-up synthetic biology is to reconstitute the machinery for binary fission, even though the precise mechanisms for regulation of the cell size have not been fully elucidated 85 . This bold aim has come into reach by the recently acquired experimental ability to reproducibly place and study cytoskeletal machinery inside vesicles 86 . There has been a significant progress in minimal divisomes, e.g., division of liposomes by bacterial Z rings 87 . However, the assembly and orchestration of several processes 88 , including complex reaction networks for positioning and geometry sensing (sizers) 89 or machinery for DNA segregation 90 , will require ingenious craftsmanship and will cover the concluding stages of cell shaping. In fact, prebiotic division should have resulted from much simpler physicochemical and intrinsic triggers, and was very likely far from being symmetric. Moreover, modern organisms still exhibit budding, hyphal growth or daughter cell formation.
Rudimental division of liposomes is often accompanied by an imbalance between volume and area and relies on some minimal "cytosol". Such examples have been demonstrated with encapsulated PEG 91 and dextran/PEG, whereby the latter led to asymmetric budding, aided by the phase-separated aqueous system 92 . Biochemically active cytosols also may lead to division -enzymatic hydrolysis of urea enabled the splitting of mixed phospholipid/fatty acid vesicles thanks to the resulting osmotic gradient and susceptibility of oleic acid to pH change 18 . In another case, osmotic deflation was induced by external enzymatic reaction, while the main driving force for parting the liposomes was phase separation of the membrane 93 . While deflation of hollow liposomes has proved as a practical trigger for division and it serves as an indispensable tool when studying membrane physics, it is difficult to ascribe the first division events solely to a floppy, relaxed membrane, provided the simultaneously required nutrient uptake and metabolism in the crowded cytosol. In fact, HeLa cells were found to increase their surface and hydrostatic pressure during mitotic rounding, which might expel water, but the concomitant osmoregulation masked any measurable changes in volume 94 .
Other bottom-up division strategies focus on the manipulation by proteins in the framework of membrane remodeling. One such artificial approach employed the adsorption of His-tagged GFP to NTA-functionalized phospholipids at low density and enabled recording morphology diagrams based on the degree of deflation (i.e. the surface-to-volume ratio) and the spontaneous curvature, whereby the curvature also affected the constriction force, necessary for neck fission 95 . This is an example how the theoretical milestones for division become attainable in a minimal system, even though GFP has been externally supplied. The physicochemical fundaments of primitive division can be further solidified by testing other membrane effectors, even if they normally exercise quite different roles in biology. One such example, facilitated by the decreasing cost and wide availability of DNA synthesis, is DNA origami as it currently enables attaining various shapes in a predictable and straightforward way 96 . Thus, DNA origami that mimicked the prominent BAR protein domains resulted in vesicle tubulation for instance 97 . In this regard, computational leaps in protein folding open new horizons for protein design 98 , which will hopefully mature to "protein origami" in the near future and will thus revolutionize structural biology.
DeepMind's AlphaFold 2 has already achieved prediction of protein structures on par with single crystal experimental methods 99  At the same time, other paradigmatic patterns are being assembled from scratch too. On the level of proteins, the reconstitution of the cell-division-related bacterial MinCDE system has yielded insights into how chemical energy, such as ATP, can be harnessed to promote spatiotemporal protein self-organization, resulting in the emergence of micron-scale pattern formation 104,105 , directional protein transport 106 and even the mechanical transformation of membranes 107 . On the level of transcription-translation, a minimal large-genome replicator, variation of the PURE system, was also able to regenerate its proteins for translation and transcription 108 , while a similar construct was chemostatically operated in a microfluidic device to sustain the activity 109 . Moreover, the ability of the former system for regeneration of essential proteins was tested in serial transfer experiments 110 , which conditions mimicked cellular reproduction in a few generations. Furthermore, the autocatalytic replication of a Φ25 bacteriophage genome was performed successfully, encapsulated in liposomes 111 . Integrating these studies in a thought experiment, along with the potentially attainable recursive self-reproduction of compartments that was discussed above, substantiates the hope for experimental verification of minimal life, which may in turn streamline the search for genesis.

Transport and energy generation at the interface
Compartmentation necessitates transport in and out to enable the continuous processing of matter, analogous to the microfluidic chemostat just mentioned above. In coacervates, this would be simply driven by the interplay of chemical equilibria and partitioning effects, but in membrane-bounded compartments, there is a barrier to be overcome. It can be anticipated that this transport was a stochastic process at the early onset of life, when various environmental or intrinsic triggers randomly caused membrane defects. Defects can also be artificially and reliably induced by electric fields in electroporation, a useful current technique for studying membrane biophysics 112 . It is also a practical tool for vesicle loading, e.g., to produce protein and genetic-material packed nano-vesicles (exosomes) which are useful in gene-therapy and an essential vehicle in cellular communication 113 115 . Provided that some degree of integrity is ensured, membrane defects could be reproduced by means that are more abiogenetically relevant -for instance by UV exposure that would degrade phospholipids. In this regard, transient permeability was speculated as a mechanism for DNA exchange between liposomes subjected to freeze-thaw cycles, as opposed to secretory fission and fusion 116 .  Energy compartmentation The evolution of selective protein pores is apparently difficult to be traced back to the early timeline of life, which in fact holds true for all proteins, but todays' engineering capabilities 123 may allow for identification of minimal motifs, aided by combinatorial screening of peptide libraries 124 . Combinatorial testing can be also applied with respect to the membrane, in order to determine key mechanical properties that influence protein folding, such as bending rigidity 125 . Synthetic chemistry can turn out to be useful for this endeavor, because it potentially allows obtaining more degrees of design freedom -an exemplary approach linked the surface pressure of different polymer/phospholipid mixtures to the distribution of potassium channels 126 . Even though the motivation behind such studies is application-driven, and these polymers have no biological relation whatsoever, the concomitant generation of data will lead to a better understanding of the required membrane properties, which can be then extrapolated to the early membranes.

Energy and complexity
The reconstitution of unspecific pores like hemolysin has proved useful in multiple instances of assembled biomimetic constructs. Cell-free expression of the toxin inside liposomes enabled prolonged synthesis of GFP due to the enhanced uptake of nutrients 127 , while in another case it facilitated the export of glucose as a chemical signal for communication with proteinosomes 11 . Reconstituted hemolysin has been also used to activate intracellular signaling thanks to Ca permeation and mechanosensitive channels 128 . The preference for this relatively simple protein pore is largely due to its facile insertion in membranes, which has even allowed for evolution of hemolysin mutants via liposome display from encapsulated translation machinery 129  Interestingly, shuttles with fully synthetic spiropyran chemistry have even enabled the light-driven generation of transmembrane proton gradients 131 to mock rhodopsins, which suggests that proton pumps might emerge from relatively simple chemistry. In this regard, light was also used for NADH oxidation by a polymeric photocatalyst through the generation of superoxide 132 (accompanying also the natural oxidation by complex I as a side reaction 133 ), while subsequent coupling with a silica-encapsulated enzyme system for cellular defense counteracted the oxidative stress 134 . This synthetic example is difficult to be conceptually linked to possible cellular energetics, as the electrons from NADH would be wasted in vain.
However, it motivates an abiotic revisiting of the reverse reaction, in line with the reported photocatalytic activity of TiO2 135 and Au 136 nanoparticles towards NAD + reduction, and synthesis of nucleoside bases 137 , within the search for primitive and decoupled pathways for carbon and "hydrogen fixation".
On the other hand, the experimental modularity and tunability of a fully functional, yet minimal construct for respiration and oxidative phosphorylation (referred to as artificial mitochondrion, comprising complex I, oxidase and ATP synthase embedded in phospholipid vesicles 138 ) might be employed towards better mechanistic understanding. For instance, it has been suggested that protons generated in the final step of respiration traverse along the membrane, as evidenced by the effect of lateral distance between bo3 oxidase and ATP synthase on the ATP synthesis rates 139 . This partially conflicted the textbook cartoon of cytoplasm acidification with a direct "kinetic" coupling instead, and underlined the importance of protein clustering 140 . The association of protons with phospholipids is known and has been already experimentally platforms for compartmentation, such as droplet-based microfluidics for the production of vesicles and coacervates or cell-free systems for protein expression. These are useful tools for studying later snapshots of abiogenesis, after life is seeded via compartmentation, and allow for addressing multiple evolutionary effects. However, the experimental reenactment of the true transition from chemistry to biology will require integrated efforts. As many of the fundamental principles of that transition are still poorly understood, a partial withdrawal from deliberate manipulation to achieve life, towards combinatorial and autonomous approaches, may allow greater chances of discovery and success.
We conclude with a conceptual comparison between a popular literature reference -Frankenstein's monster 156 -and the anthropomorphic golem as a recursively appearing theme in mythology and religion.