Liquid-liquid phase transitions in complex mixtures of proteins and other molecules produce crowded compartments supporting in vitro transcription and translation. We developed a method based on picoliter water-in-oil droplets to induce coacervation in Escherichia coli cell lysate and follow gene expression under crowded and noncrowded conditions. Coacervation creates an artificial cell-like environment in which the rate of mRNA production is increased significantly. Fits to the measured transcription rates show a two orders of magnitude larger binding constant between DNA and T7 RNA polymerase, and five to six times larger rate constant for transcription in crowded environments, strikingly similar to in vivo rates. The effect of crowding on interactions and kinetics of the fundamental machinery of gene expression has a direct impact on our understanding of biochemical networks in vivo. Moreover, our results show the intrinsic potential of cellular components to facilitate macromolecular organization into membranefree compartments by phase separation.microdroplets | macromolecular crowding P rotocells are minimal compartmentalized systems exhibiting key characteristics of cellular function, including metabolism and replication (1, 2). Lipid vesicles are considered the prototypical protocell as they can form functional microscopic spherical assemblies suited for in vitro gene expression (3, 4). Compartmentalization via lipid bilayers is considered essential for the emergence of cells (4), but there are alternative models based on liquid-liquid phase transitions that lead to the emergence of compartments (5, 6). Compartmentalization is but one characteristic, as protocells ideally also mimic the highly crowded interior of living cells, which have total macromolecule concentrations in excess of 300 g/L (7). Examples in which compartmentalization and high local concentrations are obtained concurrently, include DNA brushes (8), aqueous two-phase systems (9), and liquid coacervates (10). Phase separation or coacervation occurs in a wide range of polymer and protein solutions, often triggered by changes in temperature or salt concentration, or by the addition of coacervating agents (11). The (complex) coacervate droplets that are formed in such systems present macromolecularly crowded, aqueous, physical compartments, 1-100 μm in diameter (12). Recent work has identified similar liquid phase transitions in vivo in the formation of intracellular non-membrane-bound compartments exhibiting liquid-like properties, slowed down diffusion, and strongly interacting macromolecular components (13,14). Well-studied examples are the intracellular localization of DNA or RNA and proteins in Cajal bodies, P granules, and nucleoli (15-17), which can contain over 100 components. Such complexity has not been achieved in two-phase systems in vitro (18,19). Although the physics of coacervates is well understood, progress in their development as protocell models has stalled, because of the lack of demonstrations of complex biochemical proce...
Here, we present a platform to detect cytokine (IL-2, IFN-γ, TNF-α) secretion of single, activated T-cells in droplets over time. We use a novel droplet-based microfluidic approach to encapsulate cells in monodisperse agarose droplets together with functionalized cytokine-capture beads for subsequent binding and detection of secreted cytokines from single cells. This method allows high-throughput detection of cellular heterogeneity and maps subsets within cell populations with specific functions.
Miniaturization has been the key driver for many remarkable technological developments in recent decades. Miniaturization has now also extended into biology, thereby setting the stage for high-throughput single-cell analysis. This advancement is important because, despite detailed molecular information on individual cell subtypes, virtually no information is available on the functional capacities of individual cells. Typical in vivo animal models, as well as in vitro laboratory test tube experiments, only yield a global outcome of interactions of often millions of cells rather than providing insight into the functional contribution of individual cells. Reaction volumes of biological experiments have generally been reduced from milliliters to microliters. Tools and methods that study single-cell behavior have become increasingly important, but often do not allow for high-throughput manipulation. Recent advances in (droplet-based) microfluidics enable systematic high-throughput analyses of individual cells in a highly controlled manner. The implementation of microfluidic technologies in single-cell analysis is one of the most promising approaches that not only offers new information and high-throughput screening but also enables the creation of innovative conditions that are impractical or impossible by conventional methods. In this review, we provide a comprehensive overview of recent developments in droplet-based microfluidics for single-cell studies.
A droplet-based microfluidic reaction scheme is developed where the chemical reactants are dispensed with precise volume control into pairs of droplets. The reaction is activated by coalescing droplet pairs and fast mixing inside the coalesced droplets. Furthermore, the pre-processing of the chemical products is included in the microfluidic device. This reaction scheme allows the performing of precisely volume controlled reactions and long operation times without any clogging even if precipitates or sticky gels are formed during the reaction. Using this approach and optimizing the reaction parameters, we generate mesoporous silica microspheres from a rapid gelation optimized sol-gel synthesis route. The produced silica particles have a superior surface area of 820 m(2) g(-1) and a narrow pore radius distribution of around 2.4 nm.
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