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In this review, we summarize the strategies of inducing division in synthetic cells by using physical, chemical, and biological stimuli, and highlight the future challenges to the construction of autonomous synthetic cell division.

The challenges of light-dependent biocatalytic transformations of lipophilic substrates in aqueous media are manifold. For instance, photolability of the catalyst as well as insufficient light penetration into the reaction vessel may be further exacerbated by a heterogeneously dispersed substrate. Light penetration may be addressed by performing the reaction in continuous flow, which allows two modes of applying the catalyst: (i) heterogeneously, immobilized on a carrier, which requires light-permeable supports, or (ii) homogeneously, dissolved in the reaction mixture. Taking the light-dependent photodecarboxylation of palmitic acid catalyzed by fatty-acid photodecarboxylase from

The reaction kinetics between like-charged compounds in water is extremely slow due to Coulomb repulsions. Here, we demonstrate that by screening these interactions and, in consequence, increasing the local concentration of reactants, we boost the reactions by many orders of magnitude. The reaction between negatively charged Coenzyme A molecules accelerates ~5 million-fold using cationic micelles. That is ~104 faster kinetics than in 0.5 M NaCl, although the salt is ~106 more concentrated. Rate enhancements are not limited to micelles, as evidenced by significant catalytic effects (104–105-fold) of other highly charged species such as oligomers and polymers. We generalize the observed phenomenon by analogously speeding up a non-covalent complex formation—DNA hybridization. A theoretical analysis shows that the acceleration is correlated to the catalysts’ surface charge density in both experimental systems and enables predicting and controlling reaction rates of like-charged compounds with counter-charged species.

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Inspired by biofuel-driven dissipative self-assembly in nature, chemical fuel-driven supramolecular systems have been developed showing out-of-equilibrium functions such as transient gelation and oscillations. However, these systems suffer from undesired waste accumulation and they function only in open systems. Herein, we report non-equilibrium supramolecular polymerizations in closed chemical systems. The system is built by viologens and pyranine in the presence of hydrazine hydrate. On shaking, the viologens were quickly activated by oxidation by air followed by self-assembly of pyranine into micrometer-sized nanotubes. The self-assembled nanotubes disassembled spontaneously over time by the reduced agent, with nitrogen as the only waste product. The dissipative supramolecular polymerization could be repeated at least 6 times even in a closed system. Our mechanosensitive dissipative self-assembly process could be extended to fabricate a chiral transient supramolecular helix by introducing chiral charged small molecules into the system. More specifically, we show that shaking could induce transient fluorescence enhancement or quenching depending on substitution of viologens. Ultrasound was introduced as a specific shaking way to generate template-free reproducible patterns.

Living systems use enzymatic reaction networks to process biochemical information and make decisions in response to external or internal stimuli. Here, we present a modular and reusable platform for molecular information processing using enzymes immobilised in hydrogel beads and compartmentalised in a continuous stirred tank reactor. We demonstrate how this setup allows us to perform simple arithmetic operations, such as addition, subtraction and multiplication, using various concentrations of substrates or inhibitors as inputs and the production of a fluorescent molecule as the readout.

Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a Mycoplasma bacterium-based CFE system using lysates of the genomeminimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative Mycoplasma capricolum (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of E. coli. Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of in vitro transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNAse activity, yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned E. coli cell free extracts by degrading ribosomal RNAs, indicating that the mycoplasma cells, even the minimal cell, have a surface-associated RNAse activity. However, it is not clear which gene encodes the ribonuclease. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field. Made available in DSpace on 2022-09-14T10:05:22Z (GMT). No. of bitstreams: 1 ysac008.pdf: 1730093 bytes, checksum: 3e9cfc7a0da3bd42af172a8053fad886 (MD5) Previous issue date: 2022

Regulatory pathways inside living cells employ feed-forward architectures to fulfill essential signal processing functions that aide in the interpretation of various types of inputs through noise-filtering, fold-change detection and adaptation. Although it has been demonstrated computationally that a coherent feed-forward loop (CFFL) can function as noise filter, a property essential to decoding complex temporal signals, this motif has not been extensively characterized experimentally or integrated into larger networks. Here we use post-transcriptional regulation to implement and characterize a synthetic CFFL in an Escherichia coli cell-free transcription-translation system and build larger composite feed-forward architectures. We employ microfluidic flow reactors to probe the response of the CFFL circuit using both persistent and short, noise-like inputs and analyze the influence of different circuit components on the steady-state and dynamics of the output. We demonstrate that our synthetic CFFL implementation can reliably repress background activity compared to a reference circuit, but displays low potential as a temporal filter, and validate these findings using a computational model. Our results offer practical insight into the putative noise-filtering behavior of CFFLs and show that this motif can be used to mitigate leakage and increase the fold-change of the output of synthetic genetic circuits.

Multisomes are multicompartmental structures formed by a lipid-stabilized network of aqueous droplets, which are contained by an outer oil phase. These biomimetic structures are emerging as a versatile platform for soft matter and synthetic biology applications. While several methods for producing multisomes have been described, including microfluidic techniques, approaches for generating biocompatible, monodisperse multisomes in a reproducible manner remain challenging to implement due to low throughput and complex device fabrication. Here, we report on a robust method for the dynamically controlled generation of multisomes with controllable sizes and high monodispersity from lipid-based double emulsions. The described microfluidic approach entails the use of three different phases forming a water/oil/water (W/O/W) double emulsion stabilized by lipid layers. We employ a gradient of glycerol concentration between the inner core and outer phase to drive the directed osmosis, allowing the swelling of lamellar lipid layers resulting in the formation of small aqueous daughter droplets at the interface of the inner aqueous core. By adding increasing concentrations of glycerol to the outer aqueous phase and subsequently varying the osmotic gradient, we show that key structural parameters, including the size of the internal droplets, can be specifically controlled. Finally, we show that this approach can be used to generate multisomes encapsulating small-molecule cargo, with potential applications in synthetic biology, drug delivery, and as carriers for active materials in the food and cosmetics industries.

Cell-free protein synthesis has been widely used as a "breadboard" for design of synthetic genetic networks. However, due to a severe lack of modularity, forward engineering of genetic networks remains challenging. Here, we demonstrate how a combination of optimal experimental design and microfluidics allows us to devise dynamic cell-free gene expression experiments providing maximum information content for subsequent non-linear model identification. Importantly, we reveal that applying this methodology to a library of genetic circuits, that share common elements, further increases the information content of the data resulting in higher accuracy of model parameters. To show modularity of model parameters, we design a pulse decoder and bistable switch, and predict their behaviour both qualitatively and quantitatively. Finally, we update the parameter database and indicate that network topology affects parameter estimation accuracy. Utilizing our methodology provides us with more accurate model parameters, a necessity for forward engineering of complex genetic networks.

The ability to recognize molecular patterns is essential for the continued survival of biological organisms, allowing them to sense and respond to their immediate environment. The design of synthetic gene-based classifiers has been explored previously; however, prior strategies have focused primarily on DNA strand-displacement reactions. Here, we present a synthetic in vitro transcription and translation (TXTL)-based perceptron consisting of a weighted sum operation (WSO) coupled to a downstream thresholding function. We demonstrate the application of toehold switch riboregulators to construct a TXTL-based WSO circuit that converts DNA inputs into a GFP output, the concentration of which correlates to the input pattern and the corresponding weights. We exploit the modular nature of the WSO circuit by changing the output protein to the Escherichia coli σ28-factor, facilitating the coupling of the WSO output to a downstream reporter network. The subsequent introduction of a σ28 inhibitor enabled thresholding of the WSO output such that the expression of the downstream reporter protein occurs only when the produced σ28 exceeds this threshold. In this manner, we demonstrate a genetically implemented perceptron capable of binary classification, i.e., the expression of a single output protein only when the desired minimum number of inputs is exceeded.

Recent studies have shown that the interactions between condensates and biological membranes is of functional importance. Here, we study how the interaction between complex coacervates and liposomes as model systems can lead to membrane deformation and endocytosis. Depending on the interaction strength between coacervates and liposomes, the wetting behavior ranged from non-wetting, to partial wetting (adhesion), engulfment (endocytosis), and finally complete wetting. Endocytosis of coacervates was found to be a general phenomenon: coacervates made from a wide range of components could be taken up by liposomes. A simple theory that takes into account surface energies and coacervate sizes can explain the observed coacervate-liposome interactions. Our findings can help to better understand condensate-membrane interactions in cellular systems and provide new avenues for intracellular delivery using coacervates.

Spatiotemporal organization of distinct biological processes in cytomimetic compartments is a crucial step towards engineering functional artificial cells. Mimicking controlled bi-directional molecular communication inside artificial cells remains a considerable challenge. Here we present photoswitchable molecular transport between programmable membraneless organelle-like DNA coacervates in a synthetic microcompartment. We use droplet microfluidics to fabricate membraneless non-fusing DNA coacervates by liquid-liquid phase separation in a water-in-oil droplet, and employ the interior DNA coacervates as artificial organelles to imitate intracellular communication via photo-regulated uni- and bi-directional transfer of biomolecules. Our results highlight a promising new route to assembly of multicompartment artificial cells with functional networks.

In order to create artificial enzymatic networks capable of increasingly complex behavior, an improved methodology in understanding and controlling the kinetics of these networks is needed. Here, we introduce a Bayesian analysis method allowing for the accurate inference of enzyme kinetic parameters and determination of most likely reaction mechanisms, by combining data from different experiments and network topologies in a single probabilistic analysis framework. This Bayesian approach explicitly allows us to continuously improve our parameter estimates and behavior predictions by iteratively adding new data to our models, while automatically taking into account uncertainties introduced by the experimental setups or the chemical processes in general. We demonstrate the potential of this approach by characterizing systems of enzymes compartmentalized in beads inside flow reactors. The methods we introduce here provide a new approach to the design of increasingly complex artificial enzymatic networks, making the design of such networks more efficient, and robust against the accumulation of experimental errors.

Recapitulation of 3D multicellular tissues in vitro is of great interest to the field of tumor biology to study the integrated effect of local biochemical and biophysical signals on tumor cell migration and invasion. However, most microengineered tissues and spheroids are unable to recapitulate in vitro the complexities of 3D geometries found in vivo. Here, lithographically defined degradable alginate microniches are presented to produce free-standing tumor microtissues, with precisely controlled geometry, high viability, and allowing for high cell proliferation. The role of microtissue geometry and TGF-β signaling in tumor cell migration is further investigated. TGF-β is found to induce the expression of p-myosin II, vimentin, and YAP/TAZ nuclear localization at the periphery of the microtissue, where enhanced nuclear stiffness and orientation are also observed. Upon embedding in a collagen matrix, microtissues treated with TGF-β maintain their geometric integrity, possibly due to the higher cell tension observed around the periphery. In contrast, cells in microtissues not treated with TGF-β are highly mobile and invade the surrounding matrix rapidly, with the initial migration strongly dependent on the local geometry. The microtissues presented here are promising model systems for studying the influence of biophysical properties and soluble factors on tumor cell migration.

Systems chemistry aims to mimic the functional behavior of living systems by constructing chemical reaction networks with well-defined dynamic properties. Enzymes can play a key role in such networks, but there is currently no general and scalable route to the design and construction of enzymatic reaction networks. Here, we introduce reversible, cleavable peptide inhibitors that can link proteolytic enzymatic activity into simple network motifs. As a proof-of-principle, we show auto-activation topologies producing sigmoidal responses in enzymatic activity, explore cross-talk in minimal systems, design a simple enzymatic cascade, and introduce non-inhibiting phosphorylated peptides that can be activated using a phosphatase.

PurposeThis paper aims to describe the application of the virtual element method (VEM) to contact problems between elastic bodies.Design/methodology/approachPolygonal elements with arbitrary shape allow a stable node-to-node contact enforcement. By adaptively adjusting the polygonal mesh, this methodology is extended to problems undergoing large frictional sliding.FindingsThe virtual element is well suited for large deformation contact problems. The issue of element stability for this specific application is discussed, and the capability of the method is demonstrated by means of numerical examples.Originality/valueThis work is completely new as this is the first time, as per the authors’ knowledge, the VEM is applied to large deformation contact.

Summary To further our understanding of how biochemical information flows through cells upon external stimulation, we require single-cell multi-omics methods that concurrently map changes in (phospho)protein levels across signaling networks and the associated gene expression profiles. Here, we present quantification of RNA and intracellular epitopes by sequencing (QuRIE-seq), a droplet-based platform for single-cell RNA and intra- and extracellular (phospho)protein quantification through sequencing. We applied QuRIE-seq to quantify cell-state changes at both the signaling and the transcriptome level after 2-, 4-, 6-, 60-, and 180-min stimulation of the B cell receptor pathway in Burkitt lymphoma cells. Using the multi-omics factor analysis (MOFA+) framework, we delineated changes in single-cell (phospho)protein and gene expression patterns over multiple timescales and revealed the effect of an inhibitory drug (ibrutinib) on signaling and gene expression landscapes.

The geometry (size and shape) of gaps is a key determinant in controlling gap closure during wound healing. However, conventional methods for creating gaps result in un‐defined geometries and poorly characterized conditions (cell death factors and cell debris), which can influence the gap closure process. To overcome these limitations, a novel method to create well‐defined geometrical gaps is developed. First, smooth muscle cells (SMCs) are seeded in variously shaped micro‐containers made out of hyaluronic acid hydrogels. Cell proliferation and cell tension induce fibrous collagen production by SMCs predominantly around the edges of the micro‐containers. Upon removal of SMCs, the selectively deposited collagen results in micro‐containers with cell‐adhesive regions along the edges and walls. Fibroblasts are seeded in these micro‐containers, and upon attaching and spreading, they naturally form gaps with different geometries. The rapid proliferation of fibroblasts from the edge results in filling and closure of the gaps. It is demonstrated that gap closure rate as well as closure mechanism is strongly influenced by geometrical features, which points to an important role for cellular tension and cell proliferation in gap closure.

Ultrasensitivity is a ubiquitous emergent property of biochemical reaction networks. The design and construction of synthetic reaction networks exhibiting ultrasensitivity has been challenging, but would greatly expand the potential properties of life-like materials. Herein, we exploit a general and modular strategy to reversibly regulate the activity of enzymes using light and show how ultrasensitivity arises in simple out-of-equilibrium enzymatic systems upon incorporation of reversible photoswitchable inhibitors (PIs). Utilizing a chromophore/warhead strategy, PIs of the protease α-chymotrypsin were synthesized, which led to the discovery of inhibitors with large differences in inhibition constants (Ki) for the different photoisomers. A microfluidic flow setup was used to study enzymatic reactions under out-of-equilibrium conditions by continuous addition and removal of reagents. Upon irradiation of the continuously stirred tank reactor with different light pulse sequences, i.e., varying the pulse duration or frequency of UV and blue light irradiation, reversible switching between photoisomers resulted in ultrasensitive responses in enzymatic activity as well as frequency filtering of input signals. This general and modular strategy enables reversible and tunable control over the kinetic rates of individual enzyme-catalyzed reactions and makes a programmable linkage of enzymes to a wide range of network topologies feasible.

Current high-throughput single-cell multi-omics methods cannot concurrently map changes in (phospho)protein levels and the associated gene expression profiles. We present QuRIE-seq (Quantification of RNA and Intracellular Epitopes by sequencing) and use multi-factor omics analysis (MOFA+) to map signal transduction over multiple timescales. We demonstrate that QuRIE-seq can trace the activation of the B-cell receptor pathway at the minute and hour time-scale and provide insight into the mechanism of action of an inhibitory drug, Ibrutinib.

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Out-of-equilibrium molecular systems hold great promise as dynamic, reconfigurable matter that executes complex tasks autonomously. However, translating molecular scale dynamics into spatiotemporally controlled phenomena emerging at mesoscopic scale remains a challenge—especially if one aims at a design where the system itself maintains gradients that are required to establish spatial differentiation. Here, we demonstrate how surface tension gradients, facilitated by a linear amphiphile molecule, generate Marangoni flows that coordinate the positioning of amphiphile source and drain droplets floating at air-water interfaces. Importantly, at the same time, this amphiphile leads, via buckling instabilities in lamellar systems of said amphiphile, to the assembly of millimeter long filaments that grow from the source droplets and get absorbed at the drain droplets. Thereby, the Marangoni flows and filament organization together sustain the autonomous positioning of interconnected droplet-filament networks at the mesoscale. Our concepts provide potential for the development of non-equilibrium matter with spatiotemporal programmability., Buckling instabilities in amphiphile-based lamellar systems can lead to the formation of tubular fingers. Van der Weijden et al. show how to shepherd their growth and destination by using drain droplets that help establishing stable interconnected mesoscale droplet networks.

AFM studies on cationic polymer brushes in water (a good solvent) show that brushes in an extended conformation can be easily deformed or indented by an AFM tip. Experiments on polymer brushes in a more collapsed conformation, using methanol-water as a 'poor solvent' environment, show similar properties. Conversely, this 'soft' behaviour is dramatically different in the presence of electrolytes containing anions that are strongly coordinated to cationic groups of the polymer brush. Our initial studies in electrolyte solutions at different concentrations show that these brushes become so rigid that they cannot be indented or deformed by the AFM tip, even at high loads.

How simple chemical reactions self-assembled into complex, robust networks at the origin of life is unknown. This general problem-self-assembly of dissipative molecular networks-is also important in understanding the growth of complexity from simplicity in molecular and biomolecular systems. Here, we describe how heterogeneity in the composition of a small network of oscillatory organic reactions can sustain (rather than stop) these oscillations, when homogeneity in their composition does not. Specifically, multiple reactants in an amide-forming network sustain oscillation when the environment (here, the space velocity) changes, while homogeneous networks-those with fewer reactants-do not. Remarkably, a mixture of two reactants of different structure-neither of which produces oscillations individually-oscillates when combined. These results demonstrate that molecular heterogeneity present in mixtures of reactants can promote rather than suppress complex behaviors.

The central question addressed in this study is whether cells with different sizes have different responses to matrix stiffness. We used methacrylated hyaluronic acid (MeHA) hydrogels as the matrix to prepare an in vitro 3D microniche in which the single stem cell volume and matrix stiffness can be altered independently from each other. This simple approach enabled us to decouple the effects of matrix stiffness and cell volume in 3D microenvironments. Human mesenchymal stem cells (hMSCs) were cultured in individual 3D microniches with different volumes (2800, 3600, and 6000 μm3) and stiffnesses (5, 12, and 23 kPa). We demonstrated that cell volume affected the cellular response to matrix stiffness. When cells had an optimal volume, cells could form clear stress fibers and focal adhesions on soft, intermediate, or stiff matrix. In small cells, stress fiber formation and YAP/TAZ localization were not affected by stiffness. This study highlights the importance of considering cellular volume and substrate stiffness as important cues governing cell–matrix interactions.

Transport at the molecular scale is a prerequisite for the development of future molecular factories. Here, we have designed oligoanionic molecular sliders on polycationic tracks that exploit Brownian motion and diffusive binding to transport cargo without using a chemical fuel. The presence of the polymer tracks increases the rate of bimolecular reactions between modified sliders by over two orders of magnitude. Molecular dynamics simulations showed that the sliders not only diffuse, but also jump and hop surprisingly efficiently along polymer tracks. Inspired by acetyl-coenzyme A transporting and delivering acetyl groups in many essential biochemical processes, we developed a new and unconventional type of catalytic transport involving sliders (including coenzyme A) picking up, transporting and selectively delivering molecular cargo. Furthermore, we show that the concept of diffusive binding can also be utilized for the spatially controlled transport of chemical groups across gels. This work represents a new concept for designing functional nanosystems based on random Brownian motion. One-dimensional diffusive binding represents an important mechanism used by nature to facilitate many fundamental biochemical processes. Now, a completely synthetic system with similar capabilities has been constructed. The system was exploited to significantly speed up bimolecular reactions and to catalytically transport molecular cargo in solution and within physically separated compartments.

A droplet-based microfluidics technique is used to produce monodisperse, 80 μm collagen–gelatin beads with tunable mechanical properties in the range of 1–10 kPa after photo-crosslinking. The gel beads are porous, mechanically robust and stable in buffer, but can be degraded enzymatically. Encapsulated fibroblast cells maintain 70% viability after one-week encapsulation and preliminary results show that the degree of spreading of cells in gels is correlated with the stiffness of the material.

The reproduction of emergent behaviors in nature using reaction networks is an important objective in synthetic biology and systems chemistry. Herein, the first experimental realization of an enzymatic reaction network capable of an adaptive response is reported. The design is based on the dual activity of trypsin, which activates chymotrypsin while at the same time generating a fluorescent output from a fluorogenic substrate. Once activated, chymotrypsin counteracts the trypsin output by competing for the fluorogenic substrate and producing a non-fluorescent output. It is demonstrated that this network produces a transient fluorescent output under out-of-equilibrium conditions while the input signal persists. Importantly, in agreement with mathematical simulations, we show that optimization of the pulse-like response is an inherent trade-off between maximum amplitude and lowest residual fluorescence.

Out-of-equilibrium molecular systems hold great promise as dynamic, reconfigurable matter that executes complex tasks autonomously. However, translating molecular scale dynamics into spatiotemporally controlled phenomena at mesoscopic length scales remains a challenge. In living cells, reliable positioning processes such as the centering of the centrosome involve forces that result from dissipative self-assembly. We demonstrate how spatiotemporal positioning emerges in synthetic systems where self-assembly is coupled to molecular fluxes originating from concentration gradients. At the core of our system are millimeter long self-assembled filaments and Marangoni flows induced by non-uniform amphiphile distributions. We demonstrate how repulsive and attractive forces that are generated as filaments organize between source and drain droplets sustain autonomous positioning of dynamic assemblies at the mesoscale. Our concepts provide a new paradigm for the development of non-equilibrium matter with spatiotemporal programmability.

The origin of the incessant rhythmical heartbeat and the mechanism of muscle contraction have fascinated scientists over centuries. This short review outlines physiological explanations that were discussed in the 19th century starting with Albrecht von Haller (1708-1777), an 18th century physiologist who proposed that the heart has an intrinsic irritability. He argued that under normal conditions the inflow of blood stimulates the heart muscle to contract by mechanical touch and distension. Johannes Muller (1800-1858, physiologist in Bonn and Berlin) contended that the influence of the sympathetic nerve, specifically the activity of intracardiac ganglia, is the foremost cause of the heartbeat. Walter H. Gaskell and Theodor Engelmann (physiologists in Cambridge and Utrecht, respectively) independently criticized this neurogenic theory. They reported experimental evidence that supported the myogenic theory of the origin of the heartbeat, which has been accepted since about 1900. The concept of cardiac mechano-sensitivity, which can be traced back to A. von Haller, is currently resurging. Concerning mechanisms of contraction, Edward A. Schafer (1850-1935), histologist and physiologist in Edinburgh, described differences between cardiac and skeletal muscle and coined the term sarcomere. Based on microscopic studies of cross-striated muscle, Schafer outlined a detailed and plausible mechanism of muscle contraction in 1892. He put forward that during muscle shortening the "clear part of the muscle substance" (actin) might pass into longitudinal canals, which exist between the "sarcous elements" (myosin). His model foresaw fundamental elements of the sliding filament model, which was discovered by the Huxleys about 60 years later.

The formation of cytomimetic protocells that capture the physicochemical aspects of living cells is an important goal in bottom-up synthetic biology. Here, we recreated the crowded cytoplasm in liposome-based protocells and studied the kinetics of cell-free gene expression in these crowded containers. We found that diffusion of key components is affected not only by macromolecular crowding but also by enzymatic activity in the protocell. Surprisingly, size-dependent diffusion in crowded conditions yielded two distinct maxima for protein synthesis, reflecting the differential impact of crowding on transcription and translation. Our experimental data show, for the first time, that macromolecular crowding induces a switch from reaction to diffusion control and that this switch depends on the sizes of the macromolecules involved. These results highlight the need to control the physical environment in the design of synthetic cells.

Summary Microfluidics permit the automated manipulation of fluids at the microscale with high throughput and spatiotemporal precision, enabling the generation of large, multidimensional datasets. Machine intelligence provides powerful predictive tools with the ability to learn from data. The analysis of microfluidics-generated data via machine learning has been applied in a variety of contexts, achieving impressive results. Here, we elaborate on the potential of operating microfluidic platforms via closed-loop data-driven models by leveraging multimodal monitoring and data-acquisition instrumentation. We believe this approach will provide a robust framework for fundamental explorations in materials science and biomedicine, with implications in fields such as drug discovery, nanomaterials, in vitro organ modeling, and developmental biology. We identify challenges and propose research strategies in the context of the prediction and optimization of chemical reactions and materials syntheses and the development of the next generation of more robust and functional organs-on-chips and emerging organoids-on-chips.

Current efforts to design functional molecular systems have overlooked the importance of coupling out-of-equilibrium behaviour with changes in the environment. Here, the authors use an oscillating reaction network and demonstrate that the application of environmental forcing, in the form of periodic changes in temperature and in the inflow of the concentration of one of the network components, removes the dependency of the periodicity of this network on temperature or flow rates and enforces a stable periodicity across a wide range of conditions. Coupling a system to a dynamic environment can thus be used as a simple tool to regulate the output of a network. In addition, the authors show that coupling can also induce an increase in behavioural complexity to include quasi-periodic oscillations.

Complex systems such as ecosystems, the climate and stock markets produce emergent behaviour which is capable of undergoing dramatic change when pushed beyond a tipping point. Such complex systems display Early Warning Signals in their behaviour when they are close to a tipping point. Here we show that a complex chemical reaction network can also display early warning signals when it is in close proximity to the boundary between oscillatory and steady state concentration behaviours. We identify early warning signals using both an active sensing method, based on the recovery time of an oscillatory response after a perturbation in temperature, and a passive sensing method, based upon a change in the shape of the oscillations. The presence of the early warning signals indicates that complex, dissipative chemical networks can intrinsically sense their proximity to a boundary between behaviours.

Single-cell analysis reveals highly metabolically active circulating cells as a novel biomarker., Despite their important role in metastatic disease, no general method to detect circulating stromal cells (CStCs) exists. Here, we present the Metabolic Assay-Chip (MA-Chip) as a label-free, droplet-based microfluidic approach allowing single-cell extracellular pH measurement for the detection and isolation of highly metabolically active cells (hm-cells) from the tumor microenvironment. Single-cell mRNA-sequencing analysis of the hm-cells from metastatic prostate cancer patients revealed that approximately 10% were canonical EpCAM+ hm-CTCs, 3% were EpCAM− hm-CTCs with up-regulation of prostate-related genes, and 87% were hm-CStCs with profiles characteristic for cancer-associated fibroblasts, mesenchymal stem cells, and endothelial cells. Kaplan-Meier analysis shows that metastatic prostate cancer patients with more than five hm-cells have a significantly poorer survival probability than those with zero to five hm-cells. Thus, prevalence of hm-cells is a prognosticator of poor outcome in prostate cancer, and a potentially predictive and therapy response biomarker for agents cotargeting stromal components and preventing epithelial-to-mesenchymal transition.

Cells respond to the mechanical properties of the extracellular matrix (ECM) through formation of focal adhesions (FAs), re-organization of the actin cytoskeleton and adjustment of cell contractility. These are energy-demanding processes, but a potential causality between mechanical cues (matrix stiffness) and cellular (energy) metabolism remains largely unexplored. Here, we culture human mesenchymal stem cells (hMSCs) on stiff (20 kPa) or soft (1 kPa) substrate and demonstrate that cytoskeletal reorganization and FA formation spreading on stiff substrates lead to a drop in intracellular ATP levels, correlates with the activation of AMP-activated protein kinase (AMPK). The resulting increase in ATP levels further facilitates cell spreading and reinforces cell tension of the steady state, and coincides with nuclear localization of YAP/TAZ and Runx2. While on soft substrates (1 kPa), lowered ATP levels limit these cellular mechanoresponses. Furthermore, genetic ablation of AMPK lowered cellular ATP levels on stiff substrate and strongly reduced responses to substrate stiffness. Together, these findings reveal a hitherto unidentified relationship between energy expenditure and the cellular mechanoresponse, and point to AMPK as a key mediator of stem cell fate in response to ECM mechanics.

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The creation of adaptive matter is heavily inspired by biological systems. However, it remains challenging to design complex material responses that are governed by reaction networks, which lie at the heart of cellular complexity. The main reason for this slow progress is the lack of a general strategy to integrate reaction networks with materials. Herein we use a systematic approach to preprogram the response of a hydrogel to a trigger, in this case the enzyme trypsin, which activates a reaction network embedded within the hydrogel. A full characterization of all the kinetic rate constants in the system enabled the construction of a computational model, which predicted different hydrogel responses depending on the input concentration of the trigger. The results of the simulation are in good agreement with experimental findings. Our methodology can be used to design new, adaptive materials of which the properties are governed by reaction networks of arbitrary complexity.

A new discipline of “systems chemistry” is emerging, which aims to capture the complexity observed in natural systems within a synthetic chemical framework. Living systems rely on complex networks of chemical reactions to control the concentration of molecules in space and time. Despite the enormous complexity in biological networks, it is possible to identify network motifs that lead to functional outputs such as bistability or oscillations. To truly understand how living systems function, we need a complete understanding of how chemical reaction networks (CRNs) create function. We propose the development of a bottom-up approach to design and construct CRNs where we can follow the influence of single chemical entities on the properties of the network as a whole. Ultimately, this approach should allow us to not only understand such complex networks but also to guide and control their behavior.

The extracellular matrix consists of a complex mixture of fibrillar proteins, in which the architecture and mechanical properties of the protein fibrils vary considerably in various tissues. Here, we systematically polymerized collagen gels at different temperatures, providing substrates with tunable mechanics and defined local microarchitecture. We studied the dependence of spreading dynamics, proliferation, migration, and differentiation of human mesenchymal stem cells (hMSCs) on the fibrillar properties as compared to the bulk properties of the matrix. We found that high fiber stiffness, together with shorter fiber lengths, limited the transfer of cellular traction forces to nearby fibers. As a result, cells were not able to build up sufficient tension, which suppressed cell spreading, proliferation, and migration. Cells on such fibers also showed limited focal adhesion formation and different lineage selection preferences. In contrast, cell spreading, proliferation, and migration was always associated with fiber recruitment, long-range deformations in the collagen gel networks and an increase in collagen density around cells. Typically, cells on such substrates had a preference for osteogenic differentiation and showed higher levels of focal adhesions formation. These results contribute to a further understanding of the mechanotransduction process and to the design criteria for future biomimetic materials for tissue-engineering applications.

In recent years, there is a growing interest in designing artificial analogues of living systems, fueled not only by potential applications as 'smart micro-machines', but also by the demand for simple models that can be used to study the behavior of their more complex natural counterparts. Here, we present a facile, internally driven, experimental system comprised of fluorescently labeled colloidal silica rods of which the self-propulsion is powered by the decomposition of H2O2 catalyzed by a length-wise half Pt coating of the particles in order to study how shape anisotropy and swimming direction affect the collective behavior. We investigated the emerging structures and their time evolution for various particle concentrations in (quasi-)two dimensional systems for three aspect ratios of the rods on a single particle level using a combination of experiments and simulations. We found that the dynamic self-organization relied on a competition between self-propulsion and phoretic attractions induced by phoresis of the rods. We observed that the particle clustering behavior depends on the concentration as well as the aspect ratio of the rods. Our findings provide a more detailed understanding of dynamic self-organization of anisotropic particles and the role the propulsion direction plays in internally driven systems.

The physical distance between genes plays important roles in controlling gene expression reactions in vivo. Herein, we report the design and synthesis of a branched gene architecture in which three transcription units are integrated into one framework through assembly based on the polymerase chain reaction (PCR), together with the exploitation of these constructs as "gene compartments" for cell-free gene expression reactions, probing the impact of this physical environment on gene transcription and translation. We find that the branched gene system enhances gene expression yields, in particular at low concentrations of DNA and RNA polymerase (RNAP); furthermore, in a crowded microenvironment that mimics the intracellular microenvironment, gene expression from branched genes maintains a relatively high level. We propose that the branched gene assembly forms a membrane-free gene compartment that resembles the nucleoid of prokaryotes and enables RNAP to shuttle more efficiently between neighboring transcription units, thus enhancing gene expression efficiency. Our branched DNA architecture provides a valuable platform for studying the influence of "cellular" physical environments on biochemical reactions in simplified cell-free systems.

Environmental stimuli often lead to heterogeneous cellular responses and transcriptional output. We developed single-cell RNA and Immunodetection (RAID) to allow combined analysis of the transcriptome and intracellular (phospho-)proteins from fixed single cells. RAID successfully recapitulated differentiation-state changes at the protein and mRNA level in human keratinocytes. Furthermore, we show that differentiated keratinocytes that retain high phosphorylated FAK levels, a feature associated with stem cells, also express a selection of stem cell associated transcripts. Our data demonstrates that RAID allows investigation of heterogeneous cellular responses to environmental signals at the mRNA and phospho-proteome level.

The goal of creating a synthetic cell necessitates the development of reaction networks which will underlie all of its behaviours. Recent developments in in vitro systems, based upon both DNA and enzymes, have created networks capable of a range of behaviours e.g. information processing, adaptation and diffusive signalling. These networks are based upon reaction motifs that when combined together produce more complex behaviour. We highlight why it is inevitable that networks, based on enzymes or enzyme-like catalysts, will be required for the construction of a synthetic cell. We outline several of the challenges, including (a) timing, (b) regulation and (c) energy distribution, that must be overcome in order to transition from the simple networks we have today to much more complex networks capable of a variety of behaviours and which could find application one day within a synthetic cell.

Circulating tumor cells (CTCs) belong to a heterogeneous pool of rare cells, and a unequivocal phenotypic definition of CTC is lacking. Here, we present a definition of metabolically-altered CTC (MBA-CTCs) as CD45-negative cells with an increased extracellular acidification rate, detected with a single-cell droplet microfluidic technique. We tested the prognostic value of MBA-CTCs in 31 metastatic breast cancer patients before starting a new systemic therapy (T0) and 3&ndash, 4 weeks after (T1), comparing results with a parallel FDA-approved CellSearch (CS) approach. An increased level of MBA-CTCs was associated with: i) a shorter median PFS pre-therapy (123 days vs. 306, p &lt, 0.0001) and during therapy (139 vs. 266 days, p = 0.0009), ii) a worse OS pre-therapy (p = 0.0003, 82% survival vs. 20%) and during therapy (p = 0.0301, 67% survival vs. 38%), iii) good agreement with therapy response (kappa = 0.685). The trend of MBA-CTCs over time (combining data at T0 and T1) added information with respect to separate evaluation of T0 and T1. The combined results of the two assays (MBA and CS) increased stratification accuracy, while correlation between MBA and CS was not significant, suggesting that the two assays are detecting different CTC subsets. In conclusion, this study suggests that MBA allows detection of both EpCAM-negative and EpCAM-positive, viable and label-free CTCs, which provide clinical information apparently equivalent and complementary to CS. A further validation of proposed method and cut-offs is needed in a larger, separate study.

The compartmentalization of cell-free gene expression systems in liposomes provides an attractive route to the formation of protocells, but these models do not capture the physical (crowded) environment found in living systems. Here, we present a microfluidics-based route to produce monodisperse liposomes that can shrink almost 3 orders of magnitude without compromising their stability. We demonstrate that our strategy is compatible with cell-free gene expression and show increased protein production rates in crowded liposome protocells.