Hybrid organic-inorganic structures trigger the formation of primitive cell-like compartments.

Alkaline hydrothermal vents have become a candidate setting for the origins of life on Earth and beyond. This is due to several key features including the presence of gradients of temperature, redox potential, pH, the availability of inorganic minerals, and the existence of a network of inorganic pore spaces that could have served as primitive compartments. Chemical gardens have long been used as experimental proxies for hydrothermal vents. This paper investigates-10pc]Please note that the spelling of the following author name in the manuscript differs from the spelling provided in the article metadata: Richard J. G. Löffler. The spelling provided in the manuscript has been retained; please confirm. a set of prebiotic interactions between such inorganic structures and fatty alcohols. The integration of a medium-chain fatty alcohol, decanol, within these inorganic minerals, produced a range of emergent 3 dimensions structures at both macroscopic and microscopic scales. Fatty alcohols can be considered plausible prebiotic amphiphiles that might have assisted the formation of protocellular structures such as vesicles. The experiments presented herein show that neither chemical gardens nor decanol alone promote vesicle formation, but chemical gardens grown in the presence of decanol, which is then integrated into inorganic mineral structures, support vesicle formation. These observations suggest that the interaction of fatty alcohols and inorganic mineral structures could have played an important role in the emergence of protocells, yielding support for the evolution of living cells.

Behaviour and the Origin of Organisms.

It is common in origins of life research to view the first stages of life as the passive result of particular environmental conditions. This paper considers the alternative possibility: that the antecedents of life were already actively regulating their environment to maintain the conditions necessary for their own persistence. In support of this proposal, we describe 'viability-based behaviour': a way that simple entities can adaptively regulate their environment in response to their health, and in so doing, increase the likelihood of their survival. Drawing on empirical investigations of simple self-preserving abiological systems, we argue that these viability-based behaviours are simple enough to precede neo-Darwinian evolution. We also explain how their operation can reduce the demanding requirements that mainstream theories place upon the environment(s) in which life emerged.

Quantification of Giant Unilamellar Vesicle Fusion Products by High-Throughput Image Analysis.

Artificial cells are based on dynamic compartmentalized systems. Thus, remodeling of membrane-bound systems, such as giant unilamellar vesicles, is finding applications beyond biological studies, to engineer cell-mimicking structures. Giant unilamellar vesicle fusion is rapidly becoming an essential experimental step as artificial cells gain prominence in synthetic biology. Several techniques have been developed to accomplish this step, with varying efficiency and selectivity. To date, characterization of vesicle fusion has relied on small samples of giant vesicles, examined either manually or by fluorometric assays on suspensions of small and large unilamellar vesicles. Automation of the detection and characterization of fusion products is now necessary for the screening and optimization of these fusion protocols. To this end, we implemented a fusion assay based on fluorophore colocalization on the membranes and in the lumen of vesicles. Fluorescence colocalization was evaluated within single compartments by image segmentation with minimal user input, allowing the application of the technique to high-throughput screenings. After detection, statistical information on vesicle fluorescence and morphological properties can be summarized and visualized, assessing lipid and content transfer for each object by the correlation coefficient of different fluorescence channels. Using this tool, we report and characterize the unexpected fusogenic activity of sodium chloride on phosphatidylcholine giant vesicles. Lipid transfer in most of the vesicles could be detected after 20 h of incubation, while content exchange only occurred with additional stimuli in around 8% of vesicles.

Dynamic ordering caused by a source-sink relation between two droplets.

Two droplets composed of different chemicals, 1-decanol and liquid paraffin, floating on the water surface show characteristic co-responsive behavior. The presence of two different types of droplets in the system imposes an asymmetry that would not be possible with single droplets alone. The self-propulsion and interactions between droplets appear because surface active 1-decanol molecules provided by the source are absorbed by the paraffin sink thus generating an asymmetric surface tension gradient. This source-sink relation between droplets stabilizes and enhances the self-propulsion, and leads to a variety of dynamic structures including oscillations in the inter-droplet distance. We found that the character of time evolution also depends on the concentration of dye, Sudan Black B, initially used just to stain the decanol droplet. A simple mathematical model explains the transition between the stationary state and the oscillations as a Hopf bifurcation.

A Perfect Plastic Material for Studies on Self-Propelled Motion on the Water Surface.

We describe a novel plastic material composed of camphene, camphor, and polypropylene that seems perfectly suited for studies on self-propelled objects on the water surface. Self-motion is one of the attributes of life, and chemically propelled objects show numerous similarities with animated motion. One of important questions is the relationship between the object shape and its motility. In our previous paper, {R. Löffler et al. PCCP, 2019, 21, 24852-24856}, we presented a novel hybrid material, obtained from the solution of camphor in camphene, that allowed making objects of various shapes. This hybrid material has wax-like mechanical properties, but it has a very high tackiness. Here, we report that a small amount of polypropylene removed this undesirable feature. We investigated the properties of camphor-camphene-polypropylene plastic by performing the statistical analysis of a pill trajectory inside a Petri dish and compared them with those of camphor-camphene wax. The plastic showed the stable character of motion for over an hour-long experiment. The surface activity of objects made of plastic did not significantly depend on the weight ratios of the compounds. Such a significant increase in usefulness came from the polypropylene, which controlled the dissipation of camphor and camphene molecules.

Engineering Life: A Review of Synthetic Biology.

Synthetic biology is a field of scientific research that applies engineering principles to living organisms and living systems. It is a field that is increasing in scope with respect to organisms engineered, practical outcomes, and systems integration. There is a commercial dimension as well, where living organisms are engineered as green technologies that could offer alternatives to industrial standards in the pharmaceutical and petroleum-based chemical industries. This review attempts to provide an introduction to this field as well as a consideration of important contributions that exemplify how synthetic biology may be commensurate or even disproportionate with the complexity of living systems. The engineerability of living systems remains a difficult task, yet advancements are reported at an ever-increasing pace.

Using Imaging Flow Cytometry to Quantify and Optimize Giant Vesicle Production by Water-in-oil Emulsion Transfer Methods.

Many biologists, biochemists, and biophysicists study giant vesicles, which have a diameter of >1 µm, owing to their ease of characterization using standard optical methods. More recently, there has been interest in using giant vesicles as model systems for living cells and for the construction of artificial cells. In fact, there have been a number of reports about functionalizing giant vesicles using membrane-bound pore proteins and encapsulating biochemical reactions. Among the various methods for preparing giant vesicles, the water-in-oil emulsion transfer method is particularly well established. However, the giant vesicles prepared by this method have complex and heterogeneous properties, such as particle size and membrane structure. Here, we demonstrate the characterization of giant vesicles by imaging flow cytometry to provide quantitative and qualitative information about the vesicle products prepared by the water-in-oil emulsion transfer method. Through image-based analyses, several kinds of protocol byproducts, such as oil droplets and vesicles encapsulating no target molecules, were identified and successfully quantified. Further, the optimal agitation conditions for the water-in-oil emulsion transfer method were found from detailed analysis of imaging flow cytometry data. Our results indicate that a sonication-based water-in-oil emulsion transfer method exhibited a higher efficiency in producing giant vesicles, about 10 times or higher than that of vortex and rumble strip-based methods. It is anticipated that these approaches will be useful for fine-tuning giant vesicle production and subsequent applications.

A hybrid camphor-camphene wax material for studies on self-propelled motion.

A new material that combines the self-propelling properties of camphor with the malleability of camphene is reported. It has wax-like mechanical properties at room temperature and can be formed into required shapes. The speed of the self-propelled objects and the trajectory depend on the shape and camphor-camphene weight ratio.

Vesicle Self-Assembly of Monoalkyl Amphiphiles under the Effects of High Ionic Strength, Extreme pH, and High Temperature Environments.

Vesicles and other bilayered membranous structures can self-assemble from single hydrocarbon chain amphiphiles. Their formation and stability are highly dependent on experimental conditions such as ionic strength, pH, and temperature. The addition of divalent cations, for example, often results in the disruption of vesicles made of a single fatty acid species through amphiphile precipitation. However, membranes composed of amphiphile mixtures have been shown to be more resistant to low millimolar concentrations of divalent cations at room temperature. In this report, several mixtures of amphiphiles are examined for their propensity to self-assemble into membranous vesicular structures under extreme environmental conditions of low pH, high ionic strengths, and temperatures. In particular, mixtures of decylamine with polar cosurfactants were found to efficiently form membranes under these conditions far away from those normally supporting vesicle formation. We further examined decanoic acid/decylamine mixtures in detail. At pH 2 in low ionic strength solutions, the amphiphiles formed oily or crystalline structures; however, the introduction of salts or/and strong acids in conjunction with high temperature induced a stable vesiculation. Thus, extreme environments, such as volcanic or vent environments whose environmental conditions are known to support high chemical reactivity, could have harbored and most significantly promoted the formation of simple organic compartments that preceded cells.

Evaporation-Induced Pattern Formation of Decanol Droplets.

Pattern formation in far-from-equilibrium systems is observed in several disciplines including biology, geophysics, and reaction-diffusion chemistry, comprising both living and nonliving systems. We aim to study such nonequilibrium dynamics on the laboratory scale with materials of simple composition. We present a novel system based on a 1-decanol droplet placed in a solution of alkaline decanoate. Previously, we showed the short time scale behavior of this system, which included chemotaxis and maze solving. Here we explore long time scale dynamics of the system (several hours) when open to the environment. We observe dramatic morphological changes in the droplet including long tentacular structures, and we analyze the morphology of these structures at both the macroscopic and microscopic scales across a large range of initial conditions. Such reproducible morphological changes in simple droplets open a path to the exploration of shape-based effects in larger-scale pattern-formation studies.

Specific and Reversible DNA-Directed Self-Assembly of Modular Vesicle-Droplet Hybrid Materials.

Modular hybrid structures functionalized to assemble in a controlled manner possess diverse properties necessary for a new generation of complex materials and applications. Here, we functionalized giant unilamellar vesicles and emulsion droplets with biotinylated single-stranded DNA oligonucleotides using streptavidin as an intermediary linker to demonstrate specific and reversible DNA-directed self-assembly into vesicle-droplet hybrid structures. A low molar percentage of PEGylated phospholipids independent of the DNA-based recognition machinery at the supramolecular surface modulated the stability of the system. The reversibility of the aggregation was demonstrated by heating the hybrid structures above the melting temperature of the conjoining double-stranded DNA in the presence of excess biotin. The application of this general assembly control system to diverse multiphase soft materials provides the mechanism to assemble complex modular hybrid systems in a controllable and reversible way, which may provide an advantage where multifunctionality is a target property.

Specific and reversible DNA-directed self-assembly of oil-in-water emulsion droplets.

Higher-order structures that originate from the specific and reversible DNA-directed self-assembly of microscopic building blocks hold great promise for future technologies. Here, we functionalized biotinylated soft colloid oil-in-water emulsion droplets with biotinylated single-stranded DNA oligonucleotides using streptavidin as an intermediary linker. We show the components of this modular linking system to be stable and to induce sequence-specific aggregation of binary mixtures of emulsion droplets. Three length scales were thereby involved: nanoscale DNA base pairing linking microscopic building blocks resulted in macroscopic aggregates visible to the naked eye. The aggregation process was reversible by changing the temperature and electrolyte concentration and by the addition of competing oligonucleotides. The system was reset and reused by subsequent refunctionalization of the emulsion droplets. DNA-directed self-assembly of oil-in-water emulsion droplets, therefore, offers a solid basis for programmable and recyclable soft materials that undergo structural rearrangements on demand and that range in application from information technology to medicine.

Dynamics of chemotactic droplets in salt concentration gradients.

The chemotactic movement of decanol droplets in aqueous solutions of sodium decanoate in response to concentration gradients of NaCl has been investigated. Key parameters of the chemotactic response, namely the induction time and the migration velocity, have been evaluated as a function of the sodium decanoate concentration and the NaCl concentration gradient. The ability of the decanol droplets to migrate in concentration gradients has been demonstrated not only in a linear chemotactic assay but also in a topologically complex environment. Additionally, the ability to reverse the direction of movement repeatedly, to carry and release a chemically reactive cargo, to select a stronger concentration gradient from two options, and to initiate chemotaxis by an external temperature stimulus have been demonstrated.

Defined DNA-mediated assemblies of gene-expressing giant unilamellar vesicles.

The technological aspects of artificial vesicles as prominent cell mimics are evolving toward higher-order assemblies of functional vesicles with tissuelike architectures. Here, we demonstrate the spatially controlled DNA-directed bottom-up synthesis of complex microassemblies and macroassemblies of giant unilamellar vesicles functionalized with a basic cellular machinery to express green fluorescent protein and specified neighbor-to-neighbor interactions. We show both that the local and programmable DNA pairing rules on the nanoscale are able to direct the microscale vesicles into macroscale soft matter assemblies and that the highly sensitive gene-expression machinery remains intact and active during multiple experimental steps. An in silico model recapitulates the experiments performed in vitro and covers additional experimental setups highlighting the parameters that control the DNA-directed bottom-up synthesis of higher-order self-assembled structures. The controlled assembly of a functional vesicle matrix may be useful not only as simplified natural tissue mimics but also as artificial scaffolds that could interact and support living cells.

Survival strategies of artificial active agents.

Artificial cells can be engineered to display dynamics sharing remarkable features in common with the survival behavior of living organisms. In particular, such active systems can respond to stimuli provided by the environment and undertake specific displacements to remain out of equilibrium, e.g. by moving towards regions with higher fuel concentration. In spite of the intense experimental activity aiming at investigating this fascinating behavior, a rigorous definition and characterization of such "survival strategies" from a statistical physics perspective is still missing. In this work, we take a first step in this direction by adapting and applying to active systems the theoretical framework of Transition Path Theory, which was originally introduced to investigate rare thermally activated transitions in passive systems. We perform experiments on camphor disks navigating Petri dishes and perform simulations in the paradigmatic active Brownian particle model to show how the notions of transition probability density and committor function provide the pivotal concepts to identify survival strategies, improve modeling, and obtain and validate experimentally testable predictions. The definition of survival in these artificial systems paves the way to move beyond simple observation and to formally characterize, design and predict complex life-like behaviors.

A camphene-camphor-polymer composite material for the production of superhydrophobic absorbent microporous foams.

In a recently published paper (doi.org/10.3390/molecules26113116) on self-propelled motion of objects on the water surface, we described a novel surface-active plastic material obtained by dissolution of camphor and polypropylene in camphene at 250 [Formula: see text]C. The material has wax-like mechanical properties, can be easily formed to any moldable shape, and allows for longer and more stable self-propelled motion if compared with pure camphor or pure camphene or of a camphene-camphor wax. Here we use scanning electron microscopy to visualize and characterize the microporous structure of the solid polypropylene foam formed in the plastic for different polypropylene contents. The topology of foams remaining in the material after camphor and camphene molecules have been removed through evaporation or dissolution is similar to polypropylene foams obtained using thermally-induced phase separation. We show that the foams have a superhydrophobic surface but strongly absorb non-polar liquids, and suggest an array of potential scientific and industrial applications.

Coping with complexity: machine learning optimization of cell-free protein synthesis.

Biological systems contain complex metabolic pathways with many nonlinearities and synergies that make them difficult to predict from first principles. Protein synthesis is a canonical example of such a pathway. Here we show how cell-free protein synthesis may be improved through a series of iterated high-throughput experiments guided by a machine-learning algorithm implementing a form of evolutionary design of experiments (Evo-DoE). The algorithm predicts fruitful experiments from statistical models of the previous experimental results, combined with stochastic exploration of the experimental space. The desired experimental response, or evolutionary fitness, was defined as the yield of the target product, and new experimental conditions were discovered to have ∼ 350% greater yield than the standard. An analysis of the best experimental conditions discovered indicates that there are two distinct classes of kinetics, thus showing how our evolutionary design of experiments is capable of significant innovation, as well as gradual improvement.

Programmed vesicle fusion triggers gene expression.

The membrane properties of phospholipid vesicles can be manipulated to both regulate and initiate encapsulated biochemical reactions and networks. We present evidence for the inhibition and activation of reactions encapsulated in vesicles by the exogenous addition of charged amphiphiles. While the incorporation of cationic amphiphile exerts an inhibitory effect, complementation of additional anionic amphiphiles revitalize the reaction. We demonstrated both the simple hydrolysis reaction of ß-glucuronidase and the in vitro gene expression of this enzyme from a DNA template. Furthermore, we show that two vesicle populations decorated separately with positive and negative amphiphiles can fuse selectively to supply feeding components to initiate encapsulated reactions. This mechanism could be one of the rudimentary but effective means to regulate and maintain metabolism in dynamic artificial cell models.

Detection of association and fusion of giant vesicles using a fluorescence-activated cell sorter.

We have developed a method to evaluate the fusion process of giant vesicles using a fluorescence-activated cell sorter (FACS). Three fluorescent markers and FACS technology were used to evaluate the extent of association and fusion of giant vesicles. Two fluorescent markers encapsulated in different vesicle populations were used as association markers; when these vesicles associate, the two independent markers should be observed simultaneously in a single detection event. The quenched fluorescent marker and the dequencher, which were encapsulated in separate vesicle populations, were used as the fusion marker. When the internal aqueous solutions mix, the quenched marker is liberated by the dequencher and emits the third fluorescent signal. Although populations of pure POPC vesicles showed no detectable association or fusion, the same populations, oppositely charged by the exogenous addition of charged amphiphiles, showed up to 50% association and 30% fusion upon population analysis of 100,000 giant vesicles. Although a substantial fraction of the vesicles associated in response to a small amount of the charged amphiphiles (5% mole fraction compared to POPC alone), a larger amount of the charged amphiphiles (25%) was needed to induce vesicle fusion. The present methodology also revealed that the association and fusion of giant vesicles was dependent on size, with larger giant vesicles associating and fusing more frequently.

Autoselective transport of mammalian cells with a chemotactic droplet.

Liquid chemical droplets, as models of artificial life, when pushed away from equilibrium possess some life-like behaviors such as fission, fusion, movement and chemotaxis. Chemotaxis, directed motion in response to external gradients, is typically an important process in living systems, but certain artificial systems are also capable of this activity. Previously it was shown that droplet-based chemotactic systems when interfaced with biological systems can act as transporters to move cargo such as hydrogel alginate capsules containing living cells. Here the effectiveness of our system to transport different mammalian cell lines (H460, H1299, A549, HEK293T and HS68) was tested. It was discovered that some lung cancer cell lines release surfactants only when placed in the hydrogel capsules. These surfactants establish the interface between the encapsulated cells and the droplet and also support the chemotaxis of the droplet. Because of this, the droplet-mediated transport system is selective for living cells that produce biosurfactants. This is an example of how the integration of artificial life and biological life could be designed where the systems augment each other and function together as a unit. In this case the living system produces the surfactants that the droplet needs for cargo transport and the artificial system provides the transport for the otherwise sessile mammalian cells. Future applications of droplet-based cell handling that is able to distinguish between cells based not only on viability but cell type, developmental stage or other quantifiable traits are considered.