Steady-State Operation of a Cell-Free Genetic Band-Detection Circuit.

Pattern formation processes play a decisive role during embryogenesis and involve the precise spatial and temporal orchestration of intricate gene regulatory processes. Synthetic gene circuits modeled after their biological counterparts can be used to investigate such processes under well-controlled conditions and may, in the future, be utilized for autonomous position determination in synthetic biological materials. Here, we investigated a three-node feed-forward gene regulatory circuit in vitro that generates three distinct fluorescent outputs in response to varying concentrations of a single externally supplied morphogen. The circuit acts as a band detector for the morphogen concentration and, in a spatial context, could serve as a stripe-forming gene circuit. We simulated the behavior of the genetic circuit in the presence of a morphogen gradient using a system of ordinary differential equations and determined optimal parameters for stripe-pattern formation using an evolutionary algorithm. To analyze the subcircuits of the system, we conducted cell-free characterization experiments and finally tested the whole genetic circuit in nanoliter-scale microfluidic flow reactors that provided a continuous supply of cell extract and metabolites and allowed removal of degradation products. To make use of the widely employed promoters PLlacO-1 and PLtetO-1 in our design, we removed LacI from our bacterial cell extract by genome engineering Escherichia coli using a pORTMAGE workflow. Our results show that the band-detector works as designed when operated out of equilibrium within the flow reactors. On the other hand, subcircuits of the system and the whole circuit fail to generate the desired gene expression response when operated in a closed reactor. Our work thus underlines the importance of out-of-equilibrium operation of complex gene circuits, which cannot settle to a steady-state expression pattern within the finite lifetime of a cell-free expression system.

Complex dynamics in a synchronized cell-free genetic clock.

Complex dynamics such as period doubling and chaos occur in a wide variety of non-linear dynamical systems. In the context of biological circadian clocks, such phenomena have been previously found in computational models, but their experimental study in biological systems has been challenging. Here, we present experimental evidence of period doubling in a forced cell-free genetic oscillator operated in a microfluidic reactor, where the system is periodically perturbed by modulating the concentration of one of the oscillator components. When the external driving matches the intrinsic period, we experimentally find period doubling and quadrupling in the oscillator dynamics. Our results closely match the predictions of a theoretical model, which also suggests conditions under which our system would display chaotic dynamics. We show that detuning of the external and intrinsic period leads to more stable entrainment, suggesting a simple design principle for synchronized synthetic and natural genetic clocks.

Synthetic cell-based materials extract positional information from morphogen gradients.

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.

Programming Diffusion and Localization of DNA Signals in 3D-Printed DNA-Functionalized Hydrogels.

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.

A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a.

Point-of-care testing (POCT) in low-resource settings requires tools that can operate independently of typical laboratory infrastructure. Due to its favorable signal-to-background ratio, a wide variety of biomedical tests utilize fluorescence as a readout. However, fluorescence techniques often require expensive or complex instrumentation and can be difficult to adapt for POCT. To address this issue, we developed a pocket-sized fluorescence detector costing less than $15 that is easy to manufacture and can operate in low-resource settings. It is built from standard electronic components, including an LED and a light dependent resistor, filter foils and 3D printed parts, and reliably reaches a lower limit of detection (LOD) of ≈ 6.8 nM fluorescein, which is sufficient to follow typical biochemical reactions used in POCT applications. All assays are conducted on filter paper, which allows for a flat detector architecture to improve signal collection. We validate the device by quantifying in vitro RNA transcription and also demonstrate sequence-specific detection of target RNAs with an LOD of 3.7 nM using a Cas13a-based fluorescence assay. Cas13a is an RNA-guided, RNA-targeting CRISPR effector with promiscuous RNase activity upon recognition of its RNA target. Cas13a sensing is highly specific and adaptable and in combination with our detector represents a promising approach for nucleic acid POCT. Furthermore, our open-source device may be used in educational settings, through providing low cost instrumentation for quantitative assays or as a platform to integrate hardware, software and biochemistry concepts in the future.

Establishing Communication Between Artificial Cells.

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.

Artificial Gel-Based Organelles for Spatial Organization of Cell-Free Gene Expression Reactions.

Gel-based artificial organelles have been developed 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.