RESUMEN
The bottom-up construction of artificial cells is beneficial for understanding cell working mechanisms. The glycolysis metabolism mimicry inside artificial cells is challenging. Herein, the glycolytic pathway (Entner-Doudoroff pathway in archaea) is reconstituted inside artificial cells. The glycolytic pathway comprising glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxygluconate aldolase (KDGA) converts glucose molecules to pyruvate molecules. Inside artificial cells, pyruvate molecules are further converted into alanine with the help of alanine dehydrogenase (AlaDH) to build a metabolic pathway for synthesizing amino acid. On the other hand, the pyruvate molecules from glycolysis stimulate the living mitochondria to produce ATP inside artificial cells, which further trigger actin monomers to polymerize to form actin filaments. With the addition of methylcellulose inside the artificial cell, the actin filaments form adjacent to the inner lipid bilayer, deforming the artificial cell from a spherical shape to a spindle shape. The spindle-shaped artificial cell reverses to a spherical shape by depolymerizing the actin filament upon laser irradiation. The glycolytic pathway and its further extension to produce amino acids (or ATP) inside artificial cells pave the path to build functional artificial cells with more complicated metabolic pathways.
Asunto(s)
Aminoácidos , Células Artificiales , Glucólisis , Aminoácidos/metabolismo , Aminoácidos/química , Células Artificiales/metabolismo , Células Artificiales/químicaRESUMEN
ConspectusCreating a living system from nonliving matter is a great challenge in chemistry and biophysics. The early history of life can provide inspiration from the idea of the prebiotic "RNA World" established by ribozymes, in which all genetic and catalytic activities were executed by RNA. Such a system could be much simpler than the interdependent central dogma characterizing life today. At the same time, cooperative systems require a mechanism such as cellular compartmentalization in order to survive and evolve. Minimal cells might therefore consist of simple vesicles enclosing a prebiotic RNA metabolism.The internal volume of a vesicle is a distinctive environment due to its closed boundary, which alters diffusion and available volume for macromolecules and changes effective molecular concentrations, among other considerations. These physical effects are mechanistically distinct from chemical interactions, such as electrostatic repulsion, that might also occur between the membrane boundary and encapsulated contents. Both indirect and direct interactions between the membrane and RNA can give rise to nonintuitive, "emergent" behaviors in the model protocell system. We have been examining how encapsulation inside membrane vesicles would affect the folding and activity of entrapped RNA.Using biophysical techniques such as FRET, we characterized ribozyme folding and activity inside vesicles. Encapsulation inside model protocells generally promoted RNA folding, consistent with an excluded volume effect, independently of chemical interactions. This energetic stabilization translated into increased ribozyme activity in two different systems that were studied (hairpin ribozyme and self-aminoacylating RNAs). A particularly intriguing finding was that encapsulation could rescue the activity of mutant ribozymes, suggesting that encapsulation could affect not only folding and activity but also evolution. To study this further, we developed a high-throughput sequencing assay to measure the aminoacylation kinetics of many thousands of ribozyme variants in parallel. The results revealed an unexpected tendency for encapsulation to improve the better ribozyme variants more than worse variants. During evolution, this effect would create a tilted playing field, so to speak, that would give additional fitness gains to already-high-activity variants. According to Fisher's Fundamental Theorem of Natural Selection, the increased variance in fitness should manifest as faster evolutionary adaptation. This prediction was borne out experimentally during in vitro evolution, where we observed that the initially diverse ribozyme population converged more quickly to the most active sequences when they were encapsulated inside vesicles.The studies in this Account have expanded our understanding of emergent protocell behavior, by showing how simply entrapping an RNA inside a vesicle, which could occur spontaneously during vesicle formation, might profoundly affect the evolutionary landscape of the RNA. Because of the exponential dynamics of replication and selection, even small changes to activity and function could lead to major evolutionary consequences. By closely studying the details of minimal yet surprisingly complex protocells, we might one day trace a pathway from encapsulated RNA to a living system.
Asunto(s)
Células Artificiales , Pliegue del ARN , ARN Catalítico , ARN , Células Artificiales/química , Células Artificiales/metabolismo , ARN Catalítico/química , ARN Catalítico/metabolismo , ARN/química , ARN/metabolismo , Evolución MolecularRESUMEN
Bioprinting is an automated bioassembly method that enables the formation of human tissue-like constructs to restore or replace damaged tissues. Regardless of the employed bioprinting method, cells undergo mechanical stress that can impact their survival and function postprinting. In this study, we investigate the use of a synthetic cell-like unit, giant unilamellar vesicles (GUVs), as adjuvants of the cellular function of human cells postprinting, or in future as the complete replacement of human cells. We analyzed the impact of two nozzle-based bioprinting methods (drop-on-demand and extrusion bioprinting) on the structure, stability, and function of GUVs. We showed that over 65% of the GUVs remain intact when printing at 0.5 bar, demonstrating the potential of using GUVs as a synthetic cell source. We further increased the stability of GUVs in a cell culture medium by introducing polyethylene glycol (PEG) into the GUV lipid membrane. The presence of PEG, however, diminished the structural properties of GUVs postprinting, and reduced the interaction of GUVs with human cells. Although the design of PEG-GUVs can still be modified in future studies for better cell-GUV interactions, we demonstrated that GUVs are functional postprinting. Chlorin e6-PEG-GUVs loaded with a fluorescent dye were bioprinted, and they released the dye postprinting only upon illumination. This is a new strategy to deliver carriers, such as growth factors, drugs, nutrients, or gases, inside large bioprinted specimens on a millimeter to centimeter scale. Overall, we showed that printed GUVs can augment the functionality of manufactured human tissues.
Asunto(s)
Bioimpresión , Polietilenglicoles , Liposomas Unilamelares , Humanos , Bioimpresión/métodos , Polietilenglicoles/química , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismo , Ingeniería de Tejidos/métodos , Células Artificiales/metabolismo , Células Artificiales/química , Lípidos/químicaRESUMEN
The osmotic rupture of a cell, its osmotic lysis or cytolysis, is a phenomenon that active biological cell volume regulation mechanisms have evolved in the cell membrane to avoid. How then, at the origin of life, did the first protocells survive prior to such active processes? The pores of alkaline hydrothermal vents in the oceans form natural nanoreactors in which osmosis across a mineral membrane plays a fundamental role. Here, we discuss the dynamics of lysis and its avoidance in an abiotic system without any active mechanisms, reliant upon self-organized behaviour, similar to the first self-organized mineral membranes within which complex chemistry may have begun to evolve into metabolism. We show that such mineral nanoreactors could function as protocells without exploding because their self-organized dynamics have a large regime in parameter space where osmotic lysis does not occur and homeostasis is possible. The beginnings of Darwinian evolution in proto-biochemistry must have involved the survival of protocells that remained within such a safe regime.
Asunto(s)
Células Artificiales , Origen de la Vida , Ósmosis , Células Artificiales/metabolismo , Minerales/metabolismo , Minerales/química , Presión Osmótica , Membrana Celular/metabolismoRESUMEN
ConspectusCoacervates are droplets formed by liquid-liquid phase separation (LLPS) and are often used as model protocells-primitive cell-like compartments that could have aided the emergence of life. Their continued presence as membraneless organelles in modern cells gives further credit to their relevance. The local physicochemical environment inside coacervates is distinctly different from the surrounding dilute solution and offers an interesting microenvironment for prebiotic reactions. Coacervates can selectively take up reactants and enhance their effective concentration, stabilize products, destabilize reactants and lower transition states, and can therefore play a similar role as micellar catalysts in providing rate enhancement and selectivity in reaction outcome. Rate enhancement and selectivity must have been essential for the origins of life by enabling chemical reactions to occur at appreciable rates and overcoming competition from hydrolysis.In this Accounts, we dissect the mechanisms by which coacervate protocells can accelerate reactions and provide selectivity. These mechanisms can similarly be exploited by membraneless organelles to control cellular processes. First, coacervates can affect the local concentration of reactants and accelerate reactions by copartitioning of reactants or exclusion of a product or inhibitor. Second, the local environment inside the coacervate can change the energy landscape for reactions taking place inside the droplets. The coacervate is more apolar than the surrounding solution and often rich in charged moieties, which can affect the stability of reactants, transition states and products. The crowded nature of the droplets can favor complexation of large molecules such as ribozymes. Their locally different proton and water activity can facilitate reactions involving a (de)protonation step, condensation reactions and reactions that are sensitive to hydrolysis. Not only the coacervate core, but also the surface can accelerate reactions and provides an interesting site for chemical reactions with gradients in pH, water activity and charge. The coacervate is often rich in catalytic amino acids and can localize catalysts like divalent metal ions, leading to further rate enhancement inside the droplets. Lastly, these coacervate properties can favor certain reaction pathways, and thereby give selectivity over the reaction outcome.These mechanisms are further illustrated with a case study on ribozyme reactions inside coacervates, for which there is a fine balance between concentration and reactivity that can be tuned by the coacervate composition. Furthermore, coacervates can both catalyze ribozyme reactions and provide product selectivity, demonstrating that coacervates could have functioned as enzyme-like catalytic microcompartments at the origins of life.
Asunto(s)
Células Artificiales , Catálisis , Células Artificiales/química , Células Artificiales/metabolismo , Origen de la VidaRESUMEN
Nonlinear biomolecular interactions on membranes drive membrane remodeling crucial for biological processes including chemotaxis, cytokinesis, and endocytosis. The complexity of biomolecular interactions, their redundancy, and the importance of spatiotemporal context in membrane organization impede understanding of the physical principles governing membrane mechanics. Developing a minimal in vitro system that mimics molecular signaling and membrane remodeling while maintaining physiological fidelity poses a major challenge. Inspired by chemotaxis, we reconstructed chemically regulated actin polymerization inside vesicles, guiding membrane self-organization. An external, undirected chemical input induced directed actin polymerization and membrane deformation uncorrelated with upstream biochemical cues, suggesting symmetry breaking. A biophysical model incorporating actin dynamics and membrane mechanics proposes that uneven actin distributions cause nonlinear membrane deformations, consistent with experimental findings. This protocellular system illuminates the interplay between actin dynamics and membrane shape during symmetry breaking, offering insights into chemotaxis and other cell biological processes.
Asunto(s)
Actinas , Células Artificiales , Membrana Celular , Polimerizacion , Actinas/metabolismo , Células Artificiales/metabolismo , Células Artificiales/química , Membrana Celular/metabolismo , Quimiotaxis , Modelos BiológicosRESUMEN
A crucial step in life processes is the transfer of accurate and correct genetic material to offspring. During the construction of autonomous artificial cells, a very important step is the inheritance of genetic information in divided artificial cells. The ParMRC system, as one of the most representative systems for DNA segregation in bacteria, can be purified and reconstituted into GUVs to form artificial cells. In this study, we demonstrate that the eGFP gene is segregated into two poles by a ParM filament with ParR as the intermediate linker to bind ParM and parC-eGFP DNA in artificial cells. After the ParM filament splits, the cells are externally induced to divide into two daughter cells that contain parC-eGFP DNA by osmotic pressure and laser irradiation. Using a PURE system, we translate eGFP DNA into enhanced green fluorescent proteins in daughter cells, and bacterial plasmid segregation and inheritance are successfully mimicked in artificial cells. Our results could lead to the construction of more sophisticated artificial cells that can reproduce with genetic information.
Asunto(s)
Células Artificiales , Proteínas Fluorescentes Verdes , Plásmidos , Plásmidos/genética , Plásmidos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Células Artificiales/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Segregación Cromosómica , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMEN
Synthetic cells can be constructed from diverse molecular components, without the design constraints associated with modifying 'living' biological systems. This can be exploited to generate cells with abiotic components, creating functionalities absent in biology. One example is magnetic responsiveness, the activation and modulation of encapsulated biochemical processes using a magnetic field, which is absent from existing synthetic cell designs. This is a critical oversight, as magnetic fields are uniquely bio-orthogonal, noninvasive, and highly penetrative. Here, we address this by producing artificial magneto-responsive organelles by coupling thermoresponsive membranes with hyperthermic Fe3O4 nanoparticles and embedding them in synthetic cells. Combining these systems enables synthetic cell microreactors to be built using a nested vesicle architecture, which can respond to alternating magnetic fields through in situ enzymatic catalysis. We also demonstrate the modulation of biochemical reactions by using different magnetic field strengths and the potential to tune the system using different lipid compositions. This platform could unlock a wide range of applications for synthetic cells as programmable micromachines in biomedicine and biotechnology.
Asunto(s)
Células Artificiales , Campos Magnéticos , Células Artificiales/química , Células Artificiales/metabolismo , Nanopartículas de Magnetita/químicaRESUMEN
The Cell-Free Protein Synthesis (CFPS) system has been widely employed to facilitate the bottom-up assembly of synthetic cells. It serves as the host for the core machinery of the Central Dogma, standing as an optimal chassis for the integration and assembly of diverse artificial cellular mimicry systems. Despite its frequent use in the fabrication of synthetic cells, establishing a tailored and robust CFPS system for a specific application remains a nontrivial challenge. In this methods paper, we present a comprehensive protocol for the CFPS system, routinely employed in constructing synthetic cells. This protocol encompasses key stages in the preparation of the CFPS system, including the cell extract, template preparation, and routine expression optimization utilizing a fluorescent reporter protein. Additionally, we show representative results by encapsulating the CFPS system within various micro-compartments, such as monolayer droplets, double-emulsion vesicles, and chambers situated atop supported lipid bilayers. Finally, we elucidate the critical steps and conditions necessary for the successful assembly of these CFPS systems in distinct environments. We expect that our approach will facilitate the establishment of good working practices among various laboratories within the continuously expanding synthetic cell community, thereby accelerating progress in the field of synthetic cell development.
Asunto(s)
Células Artificiales , Sistema Libre de Células , Biosíntesis de Proteínas , Células Artificiales/química , Células Artificiales/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
Cells, the fundamental units of life, orchestrate intricate functions - motility, adaptation, replication, communication, and self-organization within tissues. Originating from spatiotemporally organized structures and machinery, coupled with information processing in signalling networks, cells embody the 'sensor-processor-actuator' paradigm. Can we glean insights from these processes to construct primitive artificial systems with life-like properties? Using de novo design approaches, what can we uncover about the evolutionary path of life? This Review discusses the strides made in crafting synthetic cells, utilizing the powerful toolbox of structural and dynamic DNA nanoscience. We describe how DNA can serve as a versatile tool for engineering entire synthetic cells or subcellular entities, and how DNA enables complex behaviour, including motility and information processing for adaptive and interactive processes. We chart future directions for DNA-empowered synthetic cells, envisioning interactive systems wherein synthetic cells communicate within communities and with living cells.
Asunto(s)
Células Artificiales , ADN , ADN/química , ADN/genética , Células Artificiales/metabolismo , Biología Sintética/métodos , Humanos , Nanotecnología/métodosRESUMEN
The RNA world hypothesis confers a central role to RNA molecules in information encoding and catalysis. Even though evidence in support of this hypothesis has accumulated from both experiments and computational modelling, the transition from an RNA world to a world where heritable genetic information is encoded in DNA remains an open question. Recent experiments show that both RNA and DNA templates can extend complementary primers using free RNA/DNA nucleotides, either non-enzymatically or in the presence of a replicase ribozyme. Guided by these experiments, we analyse protocellular evolution with an expanded set of reaction pathways made possible through the presence of DNA nucleotides. By encapsulating these reactions inside three different types of protocellular compartments, each subject to distinct modes of selection, we show how protocells containing DNA-encoded replicases in low copy numbers and replicases in high copy numbers can dominate the population. This is facilitated by a reaction that leads to auto-catalytic synthesis of replicase ribozymes from DNA templates encoding the replicase after the chance emergence of a replicase through non-enzymatic reactions. Our work unveils a pathway for the transition from an RNA world to a mixed RNA-DNA world characterized by Darwinian evolution, where DNA sequences encode heritable phenotypes.
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ADN , ARN Catalítico , ARN , ADN/genética , ADN/metabolismo , ADN/química , ARN/genética , ARN/metabolismo , ARN/química , ARN Catalítico/genética , ARN Catalítico/metabolismo , Evolución Molecular , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Células Artificiales/metabolismoRESUMEN
Here we report two novel synthetic riboswitches that respond to ASP2905 and theophylline and function in reconstituted cell-free protein synthesis (CFPS) system. We encapsulated the CFPS system as well as DNA-templated encoding reporter genes regulated by these orthogonal riboswitches inside liposomes, and achieved switchable and orthogonal control over gene expression by external stimulation with the cognate ligands.
Asunto(s)
Células Artificiales , Riboswitch , Teofilina , Teofilina/química , Células Artificiales/química , Células Artificiales/metabolismo , Liposomas/química , Regulación de la Expresión Génica , Biosíntesis de Proteínas , Sistema Libre de Células , Genes Reporteros , LigandosRESUMEN
The creation of a self-replicating synthetic cell is an essential to understand life self-replication. One method to create self-replicating artificial cells is to reconstitute the self-replication system of living organisms in vitro. In a living cell, self-replication is achieved via a system called the autonomous central dogma, a system in which central dogma-related factors are autonomously synthesized and genome replication, transcription, and translation are driven by nascent factors. Various studies to reconstitute some processes of the autonomous central dogma in vitro have been conducted. However, in vitro reconstitution of the entire autonomous central dogma system is difficult as it requires balanced expression of several related genes. Therefore, we developed a method to simultaneously quantify and optimize the in vitro expression balance of multiple genes. First, we developed a quantitative mass spectrometry method targeting genome replication-related proteins as a model of central dogma-related factors and acquired in vitro expression profiles of these genes. Additionally, we demonstrated that the in vitro expression balance of these genes can be easily optimized by adjusting the input gene ratio based on the data obtained by the developed method. This study facilitated the easy optimization of the in vitro expression balance of multiple genes. Therefore, extending the scope of this method to other central dogma-related factors will accelerate attempts of self-replicating synthetic cells creation.
Asunto(s)
Células Artificiales , Células Artificiales/metabolismo , Replicación del ADN , Espectrometría de Masas , Biología Sintética/métodos , Transcripción GenéticaRESUMEN
The design and construction of continuous flow biochemical reactors comprising immobilized biocatalysts have generated great interest in the efficient synthesis of value-added chemicals. Living cells use compartmentalization and reaction-diffusion processes for spatiotemporal regulation of biocatalytic reactions, and implementing these strategies into continuous flow reactors can offer new opportunities in reactor design and application. Herein, the fabrication of protocell-based continuous flow reactors for enzyme and whole-cell mediated biocatalysis is demonstrated. Semipermeable membranized coacervate vesicles are employed as model protocells that spontaneously sequester enzymes or accumulate living bacteria to produce embodied microreactors capable of single- or multiple-step catalytic reactions. By packing millions of the enzyme/bacteria-containing coacervate vesicles in a glass column, a facile, cost-effective, and modular methodology capable of performing oxidoreductase, peroxidase and lipolytic reactions, enzyme-mediated L-DOPA synthesis, and whole-cell glycolysis under continuous flow conditions, is demonstrated. It is shown that the protocell-nested enzymes and bacterial cells exhibit enhanced activities and stability under deleterious operating conditions compared with their non-encapsulated counterparts. These results provide a step toward the engineering of continuous flow reactors based on cell-like microscale agents and offer opportunities in the development of green and sustainable industrial bioprocessing.
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Células Artificiales , Biocatálisis , Reactores Biológicos , Células Artificiales/metabolismo , Células Artificiales/química , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Glucólisis , Enzimas/metabolismo , Enzimas/químicaRESUMEN
Lipids spontaneously assemble into vesicle-forming membranes. Such vesicles serve as compartments for even the simplest living systems. Vesicles have been extensively studied for constructing synthetic cells or as models for protocells-the cells hypothesized to have existed before life. These compartments exist almost always close to equilibrium. Life, however, exists out of equilibrium. In this work, we studied vesicle-based compartments regulated by a non-equilibrium chemical reaction network that converts activating agents. In this way, the compartments require a constant or periodic supply of activating agents to sustain themselves. Specifically, we use activating agents to condense carboxylates and phosphate esters into acyl phosphate-based lipids that form vesicles. These vesicles can only be sustained when condensing agents are present; without them, they decay. We demonstrate that the chemical reaction network can operate on prebiotic activating agents, opening the door to prebiotically plausible, self-sustainable protocells that compete for resources. In future work, such protocells should be endowed with a genotype, e.g., self-replicating RNA structures, to alter the protocell's behavior. Such protocells could enable Darwinian evolution in a prebiotically plausible chemical system.
Asunto(s)
Células Artificiales , Células Artificiales/química , Células Artificiales/metabolismo , Fosfatos/químicaRESUMEN
ATP is a universal energy currency that is essential for life. l-Arginine degradation via deamination is an elegant way to generate ATP in synthetic cells, which is currently limited by a slow l-arginine/l-ornithine exchange. We are now implementing a new antiporter with better kinetics to obtain faster ATP recycling. We use l-arginine-dependent ATP formation for the continuous synthesis and export of glycerol 3-phosphate by including glycerol kinase and the glycerol 3-phosphate/Pi antiporter. Exported glycerol 3-phosphate serves as a precursor for the biosynthesis of phospholipids in a second set of vesicles, which forms the basis for the expansion of the cell membrane. We have therefore developed an out-of-equilibrium metabolic network for ATP recycling, which has been coupled to lipid synthesis. This feeder-utilizer system serves as a proof-of-principle for the systematic buildup of synthetic cells, but the vesicles can also be used to study the individual reaction networks in confinement.
Asunto(s)
Adenosina Trifosfato , Arginina , Adenosina Trifosfato/metabolismo , Arginina/metabolismo , Células Artificiales/metabolismo , Glicerofosfatos/metabolismo , Glicerol Quinasa/metabolismo , Glicerol Quinasa/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Lípidos/biosíntesis , Fosfolípidos/metabolismo , Redes y Vías MetabólicasRESUMEN
The ability to precisely control in vitro enzymatic reactions in synthetic cells plays a crucial role in the bottom-up design of artificial cell models that can recapitulate the key cellular features and functions such as metabolism. However, integration of enzymatic reactions has been limited to bulk or microfluidic emulsions without a membrane, lacking the ability to design more sophisticated higher-order artificial cell communities for reconstituting spatiotemporal biological information at multiple length scales. Herein, droplet microfluidics is utilized to synthesize artificial cell-like polymersomes with distinct molecular permeability for spatiotemporal control of enzymatic reactions driven by external signals and fuels. The presence of a competing reverse enzymatic reaction that depletes the active substrates is shown to enable demonstration of fuel-driven formation of sub-microcompartments within polymersomes as well as realization of out-of-equilibrium systems. In addition, the different permeability characteristics of polymersome membranes are exploited to successfully construct a programmable enzymatic reaction network that mimics cellular communication within a heterogeneous cell community through selective molecular transport.
Asunto(s)
Células Artificiales , Polímeros , Células Artificiales/metabolismo , Polímeros/metabolismo , Polímeros/química , Microfluídica/métodos , Enzimas/metabolismoRESUMEN
Synthetic cell factory offers substantial advantages in economically efficient production of biofuels, chemicals, and pharmaceutical compounds. However, to create a high-performance synthetic cell factory, precise regulation of cellular material and energy flux is essential. In this context, protein components including enzymes, transcription factor-based biosensors and transporters play pivotal roles. Protein engineering aims to create novel protein variants with desired properties by modifying or designing protein sequences. This review focuses on summarizing the latest advancements of protein engineering in optimizing various aspects of synthetic cell factory, including: enhancing enzyme activity to eliminate production bottlenecks, altering enzyme selectivity to steer metabolic pathways towards desired products, modifying enzyme promiscuity to explore innovative routes, and improving the efficiency of transporters. Furthermore, the utilization of protein engineering to modify protein-based biosensors accelerates evolutionary process and optimizes the regulation of metabolic pathways. The remaining challenges and future opportunities in this field are also discussed.
Asunto(s)
Ingeniería Metabólica , Ingeniería de Proteínas , Ingeniería de Proteínas/métodos , Ingeniería Metabólica/métodos , Células Artificiales/metabolismo , Redes y Vías Metabólicas/genética , Técnicas Biosensibles , BiocombustiblesRESUMEN
The bottom-up engineering of artificial cells requires a reconfigurable cytoskeleton that can organize at distinct locations and dynamically modulate its structural and mechanical properties. Here, inspired by the vast array of actin-binding proteins and their ability to reversibly crosslink or bundle filaments, we have designed a library of peptide-DNA crosslinkers varying in length, valency and geometry. Peptide filaments conjoint through DNA hybridization give rise to tactoid-shaped bundles with tunable aspect ratios and mechanics. When confined in cell-sized water-in-oil droplets, the DNA crosslinker design guides the localization of cytoskeletal structures at the cortex or within the lumen of the synthetic cells. The tunable spatial arrangement regulates the passive diffusion of payloads within the droplets and complementary DNA handles allow for the reversible recruitment and release of payloads on and off the cytoskeleton. Heat-induced reconfiguration of peptide-DNA architectures triggers shape deformations of droplets, regulated by DNA melting temperatures. Altogether, the modular design of peptide-DNA architectures is a powerful strategy towards the bottom-up assembly of synthetic cells.
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Células Artificiales , Citoesqueleto , ADN , Péptidos , ADN/química , ADN/metabolismo , Péptidos/química , Péptidos/metabolismo , Citoesqueleto/metabolismo , Células Artificiales/química , Células Artificiales/metabolismoRESUMEN
Synthetic minimal cells are a class of bioreactors that have some, but not all, functions of live cells. Here, we report a critical step toward the development of a bottom-up minimal cell: cellular export of functional protein and RNA products. We used cell-penetrating peptide tags to translocate payloads across a synthetic cell vesicle membrane. We demonstrated efficient transport of active enzymes and transport of nucleic acid payloads by RNA-binding proteins. We investigated influence of a concentration gradient alongside other factors on the efficiency of the translocation, and we show a method to increase product accumulation in one location. We demonstrate the use of this technology to engineer molecular communication between different populations of synthetic cells, to exchange protein and nucleic acid signals. The synthetic minimal cell production and export of proteins or nucleic acids allows experimental designs that approach the complexity and relevancy of natural biological systems. A record of this paper's transparent peer review process is included in the supplemental information.