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Biomaterials composed of synthetic cells have the potential to adapt and differentiate guided by physicochemical environmental cues. Inspired by biological systems in development, which extract positional information (PI) from morphogen gradients in the presence of uncertainties, we here investigate how well synthetic cells can determine their position within a multicellular structure. To calculate PI, we created and analyzed a large number of synthetic cellular assemblies composed of emulsion droplets connected via lipid bilayer membranes. These droplets contained cell-free feedback gene circuits that responded to gradients of a genetic inducer acting as a morphogen. PI is found to be limited by gene expression noise and affected by the temporal evolution of the morphogen gradient and the cell-free expression system itself. The generation of PI can be rationalized by computational modeling of the system. We scale our approach using three-dimensional printing and demonstrate morphogen-based differentiation in larger tissue-like assemblies.

Complex non-linear dynamics such as period doubling and chaos have been previously found in computational models of the oscillatory gene networks of biological circadian clocks, but their experimental study is difficult. Here, we present experimental evidence of period doubling in a forced synthetic genetic oscillator operated in a cell-free gene expression system. To this end, an oscillatory negative feedback gene circuit is established in a microfluidic reactor, which allows continuous operation of the system over extended periods of time. We first thoroughly characterize the unperturbed oscillator and find good agreement with a four-species ODE model of the system. Guided by simulations, microfluidics is then used to periodically perturb the system by modulating the concentration of one of the oscillator components with a given amplitude and frequency. When the ratio of the external ‘zeitgeber’ period and the intrinisic period is close to 1, we experimentally find period doubling and quadrupling in the oscillator dynamics, whereas for longer zeitgeber periods, we find stable entrainment. Our theoretical model suggests favorable conditions for which the oscillator can be utilized as an externally synchronized clock, but also demonstrates that related systems could, in principle, display chaotic dynamics.

Dynamic biomaterials composed of synthetic cellular structures have the potential to adapt and functionally differentiate guided by physical and chemical cues from their environment. Inspired by developing biological systems, which efficiently extract positional information from chemical morphogen gradients in the presence of environmental uncertainties, we here investigate the analogous question: how well can a synthetic cell determine its position within a synthetic multicellular structure? In order to calculate positional information in such systems, we created and analyzed a large number of replicas of synthetic cellular assemblies, which were composed of emulsion droplets connected via lipid bilayer membranes. The droplets contained cell-free two-node feedback gene circuits that responded to gradients of a genetic inducer acting as a morphogen. We found that in our system, simple anterior-posterior differentiation is possible, but positional information is limited by gene expression noise, and is also critically affected by the temporal evolution of the morphogen gradient and the life-time of the cell-free expression system contained in the synthetic cells. Using a 3D printing approach, we demonstrate morphogen-based differentiation also in larger tissue-like assemblies.

Communication between artificial cells is essential for the realization of complex dynamical behaviors at the multi-cell level. It is also an important prerequisite for modular systems design, because it determines how spatially separated functional modules can coordinate their actions. Among others, molecular communication is required for artificial cell signaling, synchronization of cellular behaviors, computation, group-level decision-making processes and pattern formation in artificial tissues. In this review, an overview of various recent approaches to create communicating artificial cellular systems is provided. In this context, important physicochemical boundary conditions that have to be considered for the design of the communicating cells are also described, and a survey of the most striking emergent behaviors that may be achieved in such systems is given.

Additive manufacturing enables the generation of 3D structures with predefined shapes from a wide range of printable materials. However, most of the materials employed so far are static and do not provide any intrinsic programmability or pattern-forming capability. Here, a low-cost 3D bioprinting approach is developed, which is based on a commercially available extrusion printer that utilizes a DNA-functionalized bioink, which allows to combine concepts developed in dynamic DNA nanotechnology with additive patterning techniques. Hybridization between diffusing DNA signal strands and immobilized anchor strands can be used to tune diffusion properties of the signals, or to localize DNA strands within the gel in a sequence-programmable manner. Furthermore, strand displacement mechanisms can be used to direct simple pattern formation processes and to control the availability of DNA sequences at specific locations. To support printing of DNA-functionalized gel voxels at arbitrary positions, an open source python script that generates machine-readable code (GCODE) from simple vector graphics input is developed.

In biological cells, chemical processes often occur inside specialized subcellular compartments or organelles. For instance, in eukaryotes mRNA is transcribed and processed inside the nucleus, exported to the endoplasmic reticulum, and translated into the encoded protein. Inspired by this high degree of intracellular organization, we here develop gel-based artificial organelles that enable sequence-specific and programmable localization of cell-free transcription and translation reactions inside an artificial cellular system. To this end, we utilize agarose microgels covalently modified with DNA templates coding for various functions and encapsulate them into emulsion droplets. We show that RNA signals transcribed from transcription organelles can be specifically targeted to capture organelles via hybridization to the corresponding DNA addresses. We also demonstrate that mRNA molecules, produced from transcription organelles and controlled by toehold switch riboregulators, are only translated in translation organelles containing their cognate DNA triggers. Spatial confinement of transcription and translation in separate organelles is thus superficially similar to gene expression in eukaryotic cells. Combining communicating gel spheres with specialized functions opens up new possibilities for programming artificial cellular systems at the organelle level.

Position-dependent gene expression in gradients of morphogens is one of the key processes involved in cellular differentiation during development. Here, we study a simple artificial differentiation process, which is based on the diffusion of genetic inducers within one-dimensional arrangements of 50 \textgreek{m}m large water-in-oil droplets. The droplets are filled with either bacteria or cell-free gene expression systems, both equipped with genetic constructs that produce inducers or respond to them via expression of a fluorescent protein. We quantitatively study the coupled diffusion-gene expression process and demonstrate that gene expression can be made position-dependent both within bacteria-containing and cell-free droplets. By generating diffusing quorum sensing signals in situ, we also establish communication between artificial cell-free sender cells and bacterial receivers, and vice versa.