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An emerging cellular engineering method creates synthetic polymer matrices inside cells. By contrast with classical genetic, enzymatic, or radioactive techniques, this materials-based approach introduces non-natural polymers inside cells, thus modifying cellular states and functionalities. Here, we cover various materials and chemistries that have been exploited to create intracellular polymer matrices. In addition, we discuss emergent cellular properties due to the intracellular polymerization, including nonreplicating but active metabolism, maintenance of membrane integrity, and resistance to environmental stressors. We also discuss past work and future opportunities for developing and applying synthetic cells that contain intracellular polymers. The materials-based approach will usher in new applications of synthetic cells for broad biotechnological applications.

Synthetic cells can advance immunotherapy, offering innovative approaches to understanding and enhancing immune responses. This review article delves into the advancements and potential of synthetic cell technologies in immunology, emphasizing their role in understanding and manipulating immune functions. Recent progress in understanding vertebrate immune systems and the challenges posed by diseases highlight the need for innovative research methods, complementing the analysis of multidimensional datasets and genetic engineering. Synthetic immune cell engineering aims to simplify the complexity of immunological systems by reconstructing them in a controlled setting. This approach, alongside high‐throughput strategies, facilitates systematic investigations into immunity and the development of novel treatments. The article reviews synthetic cell technologies, focusing on their alignment with the three laws of immunity: universality, tolerance, and appropriateness. It explores the integration of synthetic cell modules to mimic processes such as controlled T‐cell activation, bacteria engulfment and elimination, or cellular maturation into desirable phenotypes. Together, such advancements expand the toolbox for understanding and manipulating immune functions. Synthetic cell technologies stand at the innovation crossroads in immunology, promising to illuminate fundamental immune system principles and open new avenues for research and therapy. [ABSTRACT FROM AUTHOR]

Living cells regulate many of their vital functions through dynamic, membraneless compartments that phase separate (condense) in response to different types of stimuli. In synthetic cells, responsive condensates could similarly play a crucial role in sustaining their operations. Here we use DNA nanotechnology to design and characterize artificial condensates that respond to light. These condensates form via the programmable interactions of star-shaped DNA subunits (nanostars), which are engineered to include photo-responsive protection domains. In the absence of UV irradiation, the nanostar interactions are not conducive to the formation of condensates. UV irradiation cleaves the protection domains, increases the nanostar valency and enables condensation. We demonstrate that this approach makes it possible to tune precisely the kinetics of condensate formation by dosing UV exposure time. Our experimental observations are complemented by a computational model that characterizes phase transitions of mixtures of particles of different valency, under changes in the mixture composition and bond interaction energy. In addition, we illustrate how UV activation is a useful tool to control the formation and size of DNA condensates in emulsion droplets, as a prototype organelle in a synthetic cell. This research expands our capacity to remotely control the dynamics of DNA-based components via physical stimuli and is particularly relevant to the development of minimal artificial cells and responsive biomaterials.

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. [Display omitted] [ABSTRACT FROM AUTHOR]

Synthetic cells can advance immunotherapy, offering innovative approaches to understanding and enhancing immune responses. This review article delves into the advancements and potential of synthetic cell technologies in immunology, emphasizing their role in understanding and manipulating immune functions. Recent progress in understanding vertebrate immune systems and the challenges posed by diseases highlight the need for innovative research methods, complementing the analysis of multidimensional datasets and genetic engineering. Synthetic immune cell engineering aims to simplify the complexity of immunological systems by reconstructing them in a controlled setting. This approach, alongside high‐throughput strategies, facilitates systematic investigations into immunity and the development of novel treatments. The article reviews synthetic cell technologies, focusing on their alignment with the three laws of immunity: universality, tolerance, and appropriateness. It explores the integration of synthetic cell modules to mimic processes such as controlled T‐cell activation, bacteria engulfment and elimination, or cellular maturation into desirable phenotypes. Together, such advancements expand the toolbox for understanding and manipulating immune functions. Synthetic cell technologies stand at the innovation crossroads in immunology, promising to illuminate fundamental immune system principles and open new avenues for research and therapy.

Transmembrane proteins transmit chemical signals as well as mechanical cues. The latter is often achieved by coupling to the cytoskeleton. The incorporation of fully engineerable membrane‐spanning structures for the transduction of chemical and, in particular, mechanical signals is therefore a critical aim for bottom‐up synthetic biology. Here, a membrane‐spanning DNA origami signaling units (DOSUs) is designed and mechanically coupled to DNA cytoskeletons encapsulated within giant unilamellar vesicles (GUVs). The incorporation of the DOSUs into the GUV membranes is verified and clustering upon external stimulation is achieved. Dye‐influx assays reveal that clustering increases the insertion efficiency. The transmembrane‐spanning DOSUs act as pores to allow for the transport of single‐stranded DNA into the GUVs. This is employed to trigger the reconfiguration of DNA cytoskeletons within GUVs. In addition to chemical signaling, mechanical coupling of the DOSUs to the internal DNA cytoskeletons is induced. With chemical cues from the environment, clustering of the DOSUs is induced, which triggers a symmetry break in the organization of the DNA cytoskeleton inside of the GUV. DNA‐based transmembrane structures are engineered that transduce signals without transporting the signaling molecule itself—providing a route toward signal processing and adaptive synthetic cells. [ABSTRACT FROM AUTHOR]

[Display omitted] Giant unilamellar vesicles (GUVs), cell-like synthetic micrometer size structures, assemble when thin lipid films are hydrated in aqueous solutions. Quantitative measurements of static yields and distribution of sizes of GUVs obtained from thin film hydration methods were recently reported. Dynamic data such as the time evolution of yields and distribution of sizes, however, is not known. Dynamic data can provide insights into the assembly pathway of GUVs and guidelines for choosing conditions to obtain populations with desired size distributions. We develop the 'stopped-time' technique to characterize the time evolution of the distribution of sizes and molar yields of populations of free-floating GUVs. We additionally capture high resolution time-lapse images of surface-attached GUV buds on the lipid films. We systematically study the dynamics of assembly of GUVs from three widely used thin film hydration methods, PAPYRUS (Paper-Abetted amPhiphile hYdRation in aqUeous Solutions), gentle hydration, and electroformation. We find that the molar yield versus time curves of GUVs demonstrate a characteristic sigmoidal shape, with an initial yield, a transient, and then a steady state plateau for all three methods. The population of GUVs showed a right-skewed distribution of diameters. The variance of the distributions increased with time. The systems reached steady state within 120 min. We rationalize the dynamics using the thermodynamically motivated budding and merging (BNM) model. These results further the understanding of lipid dynamics and provide for the first-time practical parameters to tailor the production of GUVs of specific sizes for applications. [ABSTRACT FROM AUTHOR]

On 25 July 2023, a workshop entitled "Molecular Communication Approaches for Wetware Artificial Life" took place as a satellite event at the international conference ALIFE 2023 (The 2023 Conference on Artificial Life). In this report, we comment on the workshop by focusing on the main theme and the motivations that led us to develop this initiative. In particular, we highlight how recent progress in synthetic biology and in the study of molecular communication from an engineering perspective can be fruitfully joined to provide a powerful platform to develop frontier research lines in "wetware" Artificial Life. The talks presented at the workshop are briefly summarized. This report is, ultimately, an opportunity to promote an emerging field that calls for collaborative efforts of scholars from multiple disciplines, from chemistry to molecular biology, from communication engineering to nanotechnology, and up to those interested in more theoretical aspects about complex artificial systems that mimic natural ones. [ABSTRACT FROM AUTHOR]

The design and construction of synthetic cells – human‐made microcompartments that mimic features of living cells – have experienced a real boom in the past decade. While many efforts have been geared toward assembling membrane‐bounded compartments, coacervate droplets produced by liquid–liquid phase separation have emerged as an alternative membrane‐free compartmentalization paradigm. Here, the dual role of coacervate droplets in synthetic cell research is discussed: encapsulated within membrane‐enclosed compartments, coacervates act as surrogates of membraneless organelles ubiquitously found in living cells; alternatively, they can be viewed as crowded cytosol‐like chassis for constructing integrated synthetic cells. After introducing key concepts of coacervation and illustrating the chemical diversity of coacervate systems, their physicochemical properties and resulting bioinspired functions are emphasized. Moving from suspensions of free floating coacervates, the two nascent roles of these droplets in synthetic cell research are highlighted: organelle‐like modules and cytosol‐like templates. Building the discussion on recent studies from the literature, the potential of coacervate droplets to assemble integrated synthetic cells capable of multiple life‐inspired functions is showcased. Future challenges that are still to be tackled in the field are finally discussed. [ABSTRACT FROM AUTHOR]

Creating an artificial cell from the bottom up is a long‐standing challenge and, while significant progress has been made, the full realization of this goal remains elusive. Arguably, one of the biggest hurdles that researchers are facing now is the assembly of different modules of cell function inside a single container. Giant unilamellar vesicles (GUVs) have emerged as a suitable container with many methods available for their production. Well‐studied swelling‐based methods offer a wide range of lipid compositions but at the expense of limited encapsulation efficiency. Emulsion‐based methods, on the other hand, excel at encapsulation but are only effective with a limited set of membrane compositions and may entrap residual additives in the lipid bilayer. Since the ultimate artificial cell will need to comply with both specific membrane and encapsulation requirements, there is still no one‐method‐fits‐all solution for GUV formation available today. This review discusses the state of the art in different GUV production methods and their compatibility with GUV requirements and operational requirements such as reproducibility and ease of use. It concludes by identifying the most pressing issues and proposes potential avenues for future research to bring us one step closer to turning artificial cells into a reality. [ABSTRACT FROM AUTHOR]

In the extant lifeforms, the self‐sustaining behaviors refer to various well‐organized biochemical reactions in spatial confinement, which rely on compartmentalization to integrate and coordinate the molecularly crowded intracellular environment and complicated reaction networks in living/synthetic cells. Therefore, the biological phenomenon of compartmentalization has become an essential theme in the field of synthetic cell engineering. Recent progress in the state‐of‐the‐art of synthetic cells has indicated that multi‐compartmentalized synthetic cells should be developed to obtain more advanced structures and functions. Herein, two ways of developing multi‐compartmentalized hierarchical systems, namely interior compartmentalization of synthetic cells (organelles) and integration of synthetic cell communities (synthetic tissues), are summarized. Examples are provided for different construction strategies employed in the above‐mentioned engineering ways, including spontaneous compartmentalization in vesicles, host–guest nesting, phase separation mediated multiphase, adhesion‐mediated assembly, programmed arrays, and 3D printing. Apart from exhibiting advanced structures and functions, synthetic cells are also applied as biomimetic materials. Finally, key challenges and future directions regarding the development of multi‐compartmentalized hierarchical systems are summarized; these are expected to lay the foundation for the creation of a "living" synthetic cell as well as provide a larger platform for developing new biomimetic materials in the future. [ABSTRACT FROM AUTHOR]

Abstract Coacervates formed by liquid‐liquid phase separation play significant roles in a variety of intracellular and extracellular biological processes. Recently, substantial efforts have been invested in creating protocells using coacervates. Microfluidic technology has rapidly gained prominence in this area due to its capability to construct monodisperse and stable coacervate droplets. This review highlights recent advancements in utilizing microfluidic devices to construct coacervate‐core‐vesicle (COV) systems. These COV systems can be employed to realize the sequestration and release of biomolecules as well as to control enzymatic reactions within the coacervate systems in a spatiotemporal manner. Lastly, we delve into the current challenges and opportunities related to the development of functional coacervate systems based on microfluidic technology.

Building synthetic multicellular systems using non‐living molecular components is a grand challenge in the field of bottom‐up synthetic biology. Towards this goal, a diverse range of chemistries have been developed to provide mechanisms of intercellular communication and methods to assemble multicellular compartments. However, building bottom‐up synthetic multicellular systems is still challenging because it requires the integration of intercellular reaction networks with compatible cellular compartment properties. In this study, we encapsulated cell‐free expression systems (CFES) expressing two quorum sensing genetic circuits into droplet interface bilayer (DIB) synthetic cells to demonstrate bidirectional communication. We further develop a method of generating custom DIB multicellular structures by acoustic liquid handling to automatically dispense the CFES droplets and show the potential for multiplexing compartmentalized gene circuits for generating heterogeneous populations of cells. Our work provides a step towards building more complex multicellular systems with intercellular communication from the bottom‐up to study and experimentally model biological multiscalar processes. [ABSTRACT FROM AUTHOR]

The recent and important advances in bottom-up synthetic biology (SB), in particular in the field of the so-called "synthetic cells" (SCs) (or "artificial cells", or "protocells"), lead us to consider the role of wetware technologies in the "Sciences of Artificial", where they constitute the third pillar, alongside the more well-known pillars hardware (robotics) and software (Artificial Intelligence, AI). In this article, it will be highlighted how wetware approaches can help to model life and cognition from a unique perspective, complementary to robotics and AI. It is suggested that, through SB, it is possible to explore novel forms of bio-inspired technologies and systems, in particular chemical AI. Furthermore, attention is paid to the concept of semantic information and its quantification, following the strategy recently introduced by Kolchinsky and Wolpert. Semantic information, in turn, is linked to the processes of generation of "meaning", interpreted here through the lens of autonomy and cognition in artificial systems, emphasizing its role in chemical ones. [ABSTRACT FROM AUTHOR]

Bottom‐up synthetic biology has reconstituted processes like adhesion, cortex formation, or division of giant lipid vesicles (GUVs), which all rely on changes in the vesicle morphology. However, oftentimes, GUV morphologies and shape transitions are described qualitatively, which makes it difficult to quantitatively compare results from different studies and to advance precision engineering. Herein, open‐source software package GeoV for the 3D reconstruction and analysis of GUV shapes from confocal microscopy z‐stacks is presented. The accuracy of the reconstruction by comparing the output for the Hausdorff distance of the surface, the curvature, and the bending energy to ground truth data for simulated shapes of different complexities is quantified. Next, GeoV on a variety of confocal microscopy datasets, including z‐stacks from spherical GUVs and GUVs deformed with DNA origami, adherent GUVs, DNA droplets, and cells, is tested. Additionally, the effect of membrane‐binding DNA origami on the vesicle shape, volume, and bending energy is quantified. It is found that osmotic deflation and attachment of DNA origami can increase the bending energy of GUVs by a factor of 10. All in all, GeoV as an open‐source software package for the quantitative analysis of confocal microscopy data for bottom‐up synthetic biology is provided. [ABSTRACT FROM AUTHOR]

On 25 July 2023, a workshop entitled “Molecular Communication Approaches for Wetware Artificial Life” took place as a satellite event at the international conference ALIFE 2023 (The 2023 Conference on Artificial Life). In this report, we comment on the workshop by focusing on the main theme and the motivations that led us to develop this initiative. In particular, we highlight how recent progress in synthetic biology and in the study of molecular communication from an engineering perspective can be fruitfully joined to provide a powerful platform to develop frontier research lines in “wetware” Artificial Life. The talks presented at the workshop are briefly summarized. This report is, ultimately, an opportunity to promote an emerging field that calls for collaborative efforts of scholars from multiple disciplines, from chemistry to molecular biology, from communication engineering to nanotechnology, and up to those interested in more theoretical aspects about complex artificial systems that mimic natural ones.

Bottom‐up synthetic biology has reconstituted processes like adhesion, cortex formation, or division of giant lipid vesicles (GUVs), which all rely on changes in the vesicle morphology. However, oftentimes, GUV morphologies and shape transitions are described qualitatively, which makes it difficult to quantitatively compare results from different studies and to advance precision engineering. Herein, open‐source software package GeoV for the 3D reconstruction and analysis of GUV shapes from confocal microscopy z‐stacks is presented. The accuracy of the reconstruction by comparing the output for the Hausdorff distance of the surface, the curvature, and the bending energy to ground truth data for simulated shapes of different complexities is quantified. Next, GeoV on a variety of confocal microscopy datasets, including z‐stacks from spherical GUVs and GUVs deformed with DNA origami, adherent GUVs, DNA droplets, and cells, is tested. Additionally, the effect of membrane‐binding DNA origami on the vesicle shape, volume, and bending energy is quantified. It is found that osmotic deflation and attachment of DNA origami can increase the bending energy of GUVs by a factor of 10. All in all, GeoV as an open‐source software package for the quantitative analysis of confocal microscopy data for bottom‐up synthetic biology is provided.

The potential role of bottom-up Synthetic Cells (SCs) in the Internet of Bio-Nano Things (IoBNT) is discussed. In particular, this perspective paper focuses on the growing interest in networks of biological and/or artificial objects at the micro- and nanoscale (cells and subcellular parts, microelectrodes, microvessels, etc.), whereby communication takes place in an unconventional manner, i.e., via chemical signaling. The resulting "molecular communication" (MC) scenario paves the way to the development of innovative technologies that have the potential to impact biotechnology, nanomedicine, and related fields. The scenario that relies on the interconnection of natural and artificial entities is briefly introduced, highlighting how Synthetic Biology (SB) plays a central role. SB allows the construction of various types of SCs that can be designed, tailored, and programmed according to specific predefined requirements. In particular, "bottom-up" SCs are briefly described by commenting on the principles of their design and fabrication and their features (in particular, the capacity to exchange chemicals with other SCs or with natural biological cells). Although bottom-up SCs still have low complexity and thus basic functionalities, here, we introduce their potential role in the IoBNT. This perspective paper aims to stimulate interest in and discussion on the presented topics. The article also includes commentaries on MC, semantic information, minimal cognition, wetware neuromorphic engineering, and chemical social robotics, with the specific potential they can bring to the IoBNT. [ABSTRACT FROM AUTHOR]

Synthetic cells, expressing proteins using cell‐free transcription‐translation (TXTL), is a technology utilized for a variety of applications, such as investigating natural gene pathways, metabolic engineering, drug development or bioinformatics. For all these purposes, the ability to precisely control gene expression is essential. Various strategies to control gene expression in TXTL have been developed; however, further advancements on gene‐specific and straightforward regulation methods are still needed. Here, we present a method of control of gene expression in TXTL using a "silencing oligo": a short oligonucleotide, designed with a particular secondary structure, that binds to the target messenger RNA. We demonstrated that silencing oligo inhibits protein expression in TXTL in a sequence‐dependent manner. We showed that silencing oligo activity is associated with RNase H activity in bacterial TXTL. To complete the gene expression control toolbox for synthetic cells, we also engineered a first transfection system. We demonstrated the transfection of various payloads, enabling the introduction of RNA and DNA of different lengths to synthetic cell liposomes. Finally, we combined the silencing oligo and the transfection technologies, demonstrating control of gene expression by transfecting silencing oligo into synthetic minimal cells. [ABSTRACT FROM AUTHOR]

The Cdv system is a protein machinery responsible for cell division in members of the TACK superphylum of archaea. The Cdv system presents many structural similarities to the eukaryotic ESCRT machinery, and it is presumed to act in a similar manner. The Cdv system has been found to be implied in a wide range of processes in the cells, such as division, vesicle budding, and virus release. The role of the CdvB paralogs is key in the division process, as became clear through recent developments in high-temperature live-cell imaging and super-resolution microscopy. In vitro experiments have shed light on the mutual interactions between CdvA, CdvC, and the various CdvB paralogs. The Cdv system is the protein machinery that performs cell division and other membrane-deforming processes in a subset of archaea. Evolutionarily, the system is closely related to the eukaryotic ESCRT machinery, with which it shares many structural and functional similarities. Since its first description 15 years ago, the understanding of the Cdv system progressed rather slowly, but recent discoveries sparked renewed interest and insights. The emerging physical picture appears to be that CdvA acts as a membrane anchor, CdvB as a scaffold that localizes division to the mid-cell position, CdvB1 and CvdB2 as the actual constriction machinery, and CdvC as the ATPase that detaches Cdv proteins from the membrane. This paper provides a comprehensive overview of the research done on Cdv and explains how this relatively understudied machinery acts to perform its cell-division function. Understanding of the Cdv system helps to better grasp the biophysics and evolution of archaea, and furthermore provides new opportunities for the bottom-up building of a divisome for synthetic cells. [ABSTRACT FROM AUTHOR]

Abstract Background Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. In addition, the yields of transcription in cell-free systems lag behind transcriptional efficiency of live cells. Most commonly used in vitro translation systems utilize T7 RNA polymerase, which is also the enzyme included in many commercial kits. Results Here we present characterization of a variant of T7 RNA polymerase promoter that acts to significantly increase the yields of gene expression within in vitro systems. We have demonstrated that T7Max increases the yield of translation in many types of commonly used in vitro protein expression systems. We also demonstrated increased protein expression yields from linear templates, allowing the use of T7Max driven expression from linear templates. Conclusions The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system.

Mimicking bacterial cell division in well‐defined cell‐free systems has the potential to elucidate the minimal set of proteins required for cytoskeletal formation, membrane constriction, and final abscission. Membrane‐anchored FtsZ polymers are often regarded as a sufficient system to realize this chain of events. By using purified FtsZ and its membrane‐binding protein FtsA or the gain‐of‐function mutant FtsA* expressed in PURE (Protein synthesis Using Reconstituted Elements) from a DNA template, it is shown in this study that cytoskeletal structures are formed, and yield constricted liposomes exhibiting various morphologies. However, the resulting buds remain attached to the parental liposome by a narrow membrane neck. No division events can be monitored even after long‐time tracking by fluorescence microscopy, nor when the osmolarity of the external solution is increased. The results provide evidence that reconstituted FtsA‐FtsZ proto‐rings coating the membrane necks are too stable to enable abscission. The prospect of combining a DNA‐encoded FtsZ system with assisting mechanisms to achieve synthetic cell division is discussed. [ABSTRACT FROM AUTHOR]

Background: Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. In addition, the yields of transcription in cell-free systems lag behind transcriptional efficiency of live cells. Most commonly used in vitro translation systems utilize T7 RNA polymerase, which is also the enzyme included in many commercial kits. Results: Here we present characterization of a variant of T7 RNA polymerase promoter that acts to significantly increase the yields of gene expression within in vitro systems. We have demonstrated that T7Max increases the yield of translation in many types of commonly used in vitro protein expression systems. We also demonstrated increased protein expression yields from linear templates, allowing the use of T7Max driven expression from linear templates. Conclusions: The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system. [ABSTRACT FROM AUTHOR]

The quest to understand life and recreate it in vitro has been undertaken through many different routes. These different approaches for experimental investigation of life aim to piece together the puzzle either by tracing life's origin or by synthesizing life‐like systems from non‐living components. Unlike efforts to define life, these experimental inquiries aim to recapture specific features of living cells, such as reproduction, self‐organization or metabolic functions that operate far from thermodynamic equilibrium. As such, these efforts have generated significant insights that shed light on crucial aspects of biological functions. For observers outside these specific research fields, it sometimes remains puzzling what properties an artificial system would need to have in order to be recognized as most similar to life. In this Perspective, we discuss properties whose realization would, in our view, allow the best possible experimental emulation of a minimal form of biological life. [ABSTRACT FROM AUTHOR]

An increased appreciation of the role of RNA dynamics in governing RNA function is ushering in a new wave of dynamic RNA synthetic biology. Here, we review recent advances in engineering dynamic RNA systems across the molecular, circuit and cellular scales for important societal-scale applications in environmental and human health, and bioproduction. For each scale, we introduce the core concepts of dynamic RNA folding and function at that scale, and then discuss technologies incorporating these concepts, covering new approaches to engineering riboswitches, ribozymes, RNA origami, RNA strand displacement circuits, biomaterials, biomolecular condensates, extracellular vesicles and synthetic cells. Considering the dynamic nature of RNA within the engineering design process promises to spark the next wave of innovation that will expand the scope and impact of RNA biotechnologies. [ABSTRACT FROM AUTHOR]

The expansion of T cells ex vivo is crucial for effective immunotherapy but currently limited by a lack of expansion approaches that closely mimic in vivo T cell activation. Taking inspiration from bottom-up synthetic biology, a new synthetic cell technology is introduced based on dispersed liquid-liquid phase-separated droplet-supported lipid bilayers (dsLBs) with tunable biochemical and biophysical characteristics, as artificial antigen presenting cells (aAPCs) for ex vivo T cell expansion. These findings obtained with the dsLB technology reveal three key insights: first, introducing laterally mobile stimulatory ligands on soft aAPCs promotes expansion of IL-4/IL-10 secreting regulatory CD8 + T cells, with a PD-1 negative phenotype, less prone to immune suppression. Second, it is demonstrated that lateral ligand mobility can mask differential T cell activation observed on substrates of varying stiffness. Third, dsLBs are applied to reveal a mechanosensitive component in bispecific Her2/CD3 T cell engager-mediated T cell activation. Based on these three insights, lateral ligand mobility, alongside receptor- and mechanosignaling, is proposed to be considered as a third crucial dimension for the design of ex vivo T cell expansion technologies., (© 2024 The Authors. Small published by Wiley‐VCH GmbH.)

Microfluidics plays a pivotal role in organ-on-chip technologies and in the study of synthetic cells, especially in the development and analysis of artificial cell models. However, approaches that use synthetic cells as integral functional components for microfluidic systems to shape the microenvironment of natural living cells cultured on-chip are not explored. Here, colloidosome-based synthetic cells are integrated into 3D microfluidic devices, pioneering the concept of synthetic cell-based microenvironments for organs-on-chip. Methods are devised to create dense and stable networks of silica colloidosomes, enveloped by supported lipid bilayers, within microfluidic channels. These networks promote receptor-ligand interactions with on-chip cultured cells. Furthermore, a technique is introduced for the controlled release of growth factors from the synthetic cells into the channels, using a calcium alginate-based hydrogel formation within the colloidosomes. To demonstrate the potential of the technology, a modular plug-and-play lymph-node-on-a-chip prototype that guides the expansion of primary human T cells by stimulating receptor ligands on the T cells and modulating their cytokine environment is presented. This integration of synthetic cells into microfluidic systems offers a new direction for organ-on-chip technologies and suggests further avenues for exploration in potential therapeutic applications., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

The potential role of bottom-up Synthetic Cells (SCs) in the Internet of Bio-Nano Things (IoBNT) is discussed. In particular, this perspective paper focuses on the growing interest in networks of biological and/or artificial objects at the micro- and nanoscale (cells and subcellular parts, microelectrodes, microvessels, etc.), whereby communication takes place in an unconventional manner, i.e., via chemical signaling. The resulting “molecular communication” (MC) scenario paves the way to the development of innovative technologies that have the potential to impact biotechnology, nanomedicine, and related fields. The scenario that relies on the interconnection of natural and artificial entities is briefly introduced, highlighting how Synthetic Biology (SB) plays a central role. SB allows the construction of various types of SCs that can be designed, tailored, and programmed according to specific predefined requirements. In particular, “bottom-up” SCs are briefly described by commenting on the principles of their design and fabrication and their features (in particular, the capacity to exchange chemicals with other SCs or with natural biological cells). Although bottom-up SCs still have low complexity and thus basic functionalities, here, we introduce their potential role in the IoBNT. This perspective paper aims to stimulate interest in and discussion on the presented topics. The article also includes commentaries on MC, semantic information, minimal cognition, wetware neuromorphic engineering, and chemical social robotics, with the specific potential they can bring to the IoBNT.

Giant polymersomes are versatile and stable biomimetic compartments that are ideal for building cell‐like systems. However, the transport of hydrophilic molecules across the membrane, which controls the function of cell‐like systems, is limited by the low permeability of polymeric bilayers. Therefore, mechanisms to control the permeability of polymersomes are necessary to create functional cell‐like systems. Here, we describe the design of giant polymersomes equipped with spiropyran‐based permeability modulators. Photo‐isomerization of the modulators leads to perturbation of the polymer membrane, resulting in increased permeability. The photoactivated polymersomes were used to construct two cell‐like systems controlled by light‐activated transport of hydrophilic molecules. First, we designed an enzymatic micro‐reactor activated by light irradiation. Second, we constructed a hybrid coacervate‐in‐polymersome system that mimics the adaptive formation of biological condensates in cells. [ABSTRACT FROM AUTHOR]

Various models of synthetic cells have been developed as researchers have sought to explore the origins of life. Based on the fact that structural complexity is the foundation of higher‐order functions, this review focuses on hierarchical structures in synthetic cell models that are inspired by living systems, in which macromolecules are the dominant participants. The underlying advantages and functions provided by biomimetic higher‐order structures are discussed from four perspectives, including hierarchical structures in membranes, in the composite construction of membrane‐coated artificial cytoplasm, in organelle‐like subcellular compartments, as well as in synthetic cell–cell assembled synthetic tissues. In parallel, various feasible driving forces and approaches for the fabrication of such higher‐order structures are showcased. Furthermore, both the implemented and potential applications of biomimetic systems, bottom‐up biosynthesis, biomedical tissue engineering, and disease therapy are highlighted. This thriving field is gradually narrowing the gap between fundamental research and applied science. [ABSTRACT FROM AUTHOR]

Advances in artificial/synthetic cells have drawn a new era of nanobiotechnology, which have shown broad prospects in biomedical applications. The rational nanoengineering of synthetic cells that can closely substitute the systematic biological functions of cells is a next grand challenge. Here, a genetically encoded synthetic beta cell, which can sense hyperglycemic conditions to initiate programmed biosynthesis and secretion of insulin is reported. By encapsulating different metal–organic framework‐based artificial organelles with distinctive bifunctionalities, the synthetic cell can undergo programmed, sequential subcellular events, including glucose sensing, initiation of insulin gene transcription and translation, and finally excretion of functional insulin, under hyperglycemic conditions. Glucose uptake assay suggests that the insulin produced by the synthetic cells can successfully promote glucose uptake into mammalian cells. The construction of a higher‐order cell cluster by ligand‐mediated super‐assembly of the synthetic cells is further demonstrated. Such a robust and smart synthetic system that closely mimics the cellular activities of beta cells in response to glucose levels is promising for improving clinical outcomes in diabetes treatment. [ABSTRACT FROM AUTHOR]

The regulation of protein uptake and secretion is crucial for (inter)cellular signaling. Mimicking these molecular events is essential when engineering synthetic cellular systems. A first step towards achieving this goal is obtaining control over the uptake and release of proteins from synthetic cells in response to an external trigger. Herein, we have developed an artificial cell that sequesters and releases proteinaceous cargo upon addition of a coded chemical signal: single‐stranded DNA oligos (ssDNA) were employed to independently control the localization of a set of three different ssDNA‐modified proteins. The molecular coded signal allows for multiple iterations of triggered uptake and release, regulation of the amount and rate of protein release and the sequential release of the three different proteins. This signaling concept was furthermore used to directionally transfer a protein between two artificial cell populations, providing novel directions for engineering lifelike communication pathways inside higher order (proto)cellular structures. [ABSTRACT FROM AUTHOR]

Synthetic cells are engineered vesicles that can mimic one or more salient features of life. These features include directed localization, sense‐and‐respond behavior, gene expression, metabolism, and high stability. In nanomedicine, many of these features are desirable capabilities of drug delivery vehicles but are difficult to engineer. In this focus article, we discuss where synthetic cells offer unique advantages over nanoparticle and living cell therapies. We review progress in the engineering of the above life‐like behaviors and how they are deployed in nanomedicine. Finally, we assess key challenges synthetic cells face before being deployed as drugs and suggest ways to overcome these challenges. This article is categorized under:Therapeutic Approaches and Drug Discovery > Emerging TechnologiesBiology‐Inspired Nanomaterials > Lipid‐Based Structures [ABSTRACT FROM AUTHOR]