Controlling liquid-liquid phase behaviour with an active fluid.

Demixing binary liquids is a ubiquitous transition explained using a well-established thermodynamic formalism that requires the equality of intensive thermodynamics parameters across phase boundaries. Demixing transitions also occur when binary fluid mixtures are driven away from equilibrium, but predicting and designing such out-of-equilibrium transitions remains a challenge. Here we study the liquid-liquid phase separation of attractive DNA nanostars driven away from equilibrium using a microtubule-based active fluid. We find that activity lowers the critical temperature and narrows the range of coexistence concentrations, but only in the presence of mechanical bonds between the liquid droplets and reconfiguring active fluid. Similar behaviours are observed in numerical simulations, suggesting that the activity suppression of the critical point is a generic feature of active liquid-liquid phase separation. Our work describes a versatile platform for building soft active materials with feedback control and providing an insight into self-organization in cell biology.

Assembling Microtubule-Based Active Matter.

Studied for more than a century, equilibrium liquid crystals provided insight into the properties of ordered materials, and led to commonplace applications such as display technology. Active nematics are a new class of liquid crystal materials that are driven out of equilibrium by continuous motion of the constituent anisotropic units. A versatile experimental realization of active nematic liquid crystals is based on rod-like cytoskeletal filaments that are driven out of equilibrium by molecular motors. We describe protocols for assembling microtubule-kinesin based active nematic liquid crystals and associated isotropic fluids. We describe the purification of each protein and the assembly process of a two-dimensional active nematic on a water-oil interface. Finally, we show examples of nematic formation and describe methods for quantifying their non-equilibrium dynamics.

Multiscale Microtubule Dynamics in Active Nematics.

In microtubule-based active nematics, motor-driven extensile motion of microtubule bundles powers chaotic large-scale dynamics. We quantify the interfilament sliding motion both in isolated bundles and in a dense active nematic. The extension speed of an isolated microtubule pair is comparable to the molecular motor stepping speed. In contrast, the net extension in dense 2D active nematics is significantly slower; the interfilament sliding speeds are widely distributed about the average and the filaments exhibit both contractile and extensile relative motion. These measurements highlight the challenge of connecting the extension rate of isolated bundles to the multimotor and multifilament interactions present in a dense 2D active nematic. They also provide quantitative data that is essential for building multiscale models.

Active liquid crystals powered by force-sensing DNA-motor clusters.

Cytoskeletal active nematics exhibit striking nonequilibrium dynamics that are powered by energy-consuming molecular motors. To gain insight into the structure and mechanics of these materials, we design programmable clusters in which kinesin motors are linked by a double-stranded DNA linker. The efficiency by which DNA-based clusters power active nematics depends on both the stepping dynamics of the kinesin motors and the chemical structure of the polymeric linker. Fluorescence anisotropy measurements reveal that the motor clusters, like filamentous microtubules, exhibit local nematic order. The properties of the DNA linker enable the design of force-sensing clusters. When the load across the linker exceeds a critical threshold, the clusters fall apart, ceasing to generate active stresses and slowing the system dynamics. Fluorescence readout reveals the fraction of bound clusters that generate interfilament sliding. In turn, this yields the average load experienced by the kinesin motors as they step along the microtubules. DNA-motor clusters provide a foundation for understanding the molecular mechanism by which nanoscale molecular motors collectively generate mesoscopic active stresses, which in turn power macroscale nonequilibrium dynamics of active nematics.

Boundary-Free Ribosome Compartmentalization by Gene Expression on a Surface.

The design of artificial cell models based on minimal surface-bound transcription-translation reactions aims to mimic the compartmentalization facilitated by organelles and inner interfaces in living cells. Dense DNA brushes as localized sources of RNA and proteins serve as synthetic operons that have recently proven useful for the autonomous synthesis and assembly of cellular machines. Here, we studied ribosome compartmentalization in a minimal gene-expression reaction on a surface in contact with a macroscopic reservoir. We first observed the accumulation and colocalization of RNA polymerases, ribosomes, nascent RNAs and proteins, in dense DNA brushes using evanescent field fluorescence, showing transcription-translation coupling in the brush. Fluorescence recovery after photobleaching showed that ribosomes engaged in translation in the brush had a 4-fold slower diffusion constant. In addition, ribosomes in the brush had over a 10-fold higher local concentration relative to free ribosomes, creating a boundary-free functional ribosome-rich compartment. To decouple translation from transcription, we immobilized dense phases of ribosomes next to DNA brushes. We demonstrated that immobilized ribosomes were capable of protein synthesis, forming 2D subcompartments of active ribosome patterns induced and regulated by DNA brush layout of coding and inhibitory genes. Localizing additional molecular components on the surface will further compartmentalize gene-expression reactions.

Electric-Field Manipulation of a Compartmentalized Cell-Free Gene Expression Reaction.

Direct electric-field manipulation of gene expression reactions would simplify the design of biochemical networks by replacing complex biomolecular interactions with push-button operations. Here, we applied a localized electric field gradient at megahertz frequency to manipulate a cell-free gene-expression reaction in a DNA compartment on a chip. We broke the spatial symmetry of a homogeneous reaction in the compartment by creating a trap for macromolecules in a region of maximal field intensity localized 50 µm from immobilized DNA. Free of biochemical regulation, we demonstrated protein synthesis oscillations by on/off switching of the electric field. In response to the field, ribosomes, RNA polymerases, and nascent RNA and proteins accumulated in the trap, and were then depleted from the DNA region where gene expression occurred. The resulting reduction in the rate of protein synthesis recovered back to steady-state when the field was off. The combination of electric field with compartmentalized cell-free gene expression reactions creates a simple, label-free approach for controlling biomolecules in space and time, opening possibilities for hybrid biological systems with a bioelectronic interface based on minimal biological parts design.

Synchrony and pattern formation of coupled genetic oscillators on a chip of artificial cells.

Understanding how biochemical networks lead to large-scale nonequilibrium self-organization and pattern formation in life is a major challenge, with important implications for the design of programmable synthetic systems. Here, we assembled cell-free genetic oscillators in a spatially distributed system of on-chip DNA compartments as artificial cells, and measured reaction-diffusion dynamics at the single-cell level up to the multicell scale. Using a cell-free gene network we programmed molecular interactions that control the frequency of oscillations, population variability, and dynamical stability. We observed frequency entrainment, synchronized oscillatory reactions and pattern formation in space, as manifestation of collective behavior. The transition to synchrony occurs as the local coupling between compartments strengthens. Spatiotemporal oscillations are induced either by a concentration gradient of a diffusible signal, or by spontaneous symmetry breaking close to a transition from oscillatory to nonoscillatory dynamics. This work offers design principles for programmable biochemical reactions with potential applications to autonomous sensing, distributed computing, and biomedical diagnostics.

Progress in programming spatiotemporal patterns and machine-assembly in cell-free protein expression systems.

Building biological systems outside the cell is an emerging interdisciplinary research field aimed to study design principles, and to emulate biological functions for technology. Reconstructing programmable cellular functions, from assembly of protein/nucleic-acid machines to spatially distributed systems, requires implementing minimal systems of molecular interactions encoded in genes, source-sink protein expression dynamics, and materials platforms for reaction-diffusion scenarios. Here, we first review how molecular turnover mechanisms, combined with nonlinear interactions and feedback in cell-free gene networks enable programmable dynamic expression patterns in various compartments. We then describe recent work on spatially distributed protein expression reactions. Finally, we discuss progress and challenges in the study of programmable protein/nucleic-acid complexes.

Synthetic biology. Programmable on-chip DNA compartments as artificial cells.

The assembly of artificial cells capable of executing synthetic DNA programs has been an important goal for basic research and biotechnology. We assembled two-dimensional DNA compartments fabricated in silicon as artificial cells capable of metabolism, programmable protein synthesis, and communication. Metabolism is maintained by continuous diffusion of nutrients and products through a thin capillary, connecting protein synthesis in the DNA compartment with the environment. We programmed protein expression cycles, autoregulated protein levels, and a signaling expression gradient, equivalent to a morphogen, in an array of interconnected compartments at the scale of an embryo. Gene expression in the DNA compartment reveals a rich, dynamic system that is controlled by geometry, offering a means for studying biological networks outside a living cell.