Synthetic Biology in Aqueous Compartments at the Micro- and Nanoscale
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Synthetic Biology in Aqueous Compartments at the Micro- and Nanoscale J. Boreyko,1 P. Caveney,2,3 S. L. Norred,2,3 C. Chin,2,3 S.T. Retterer,2,3,4 M.L. Simpson,2,3 C.P. Collier2,3 1
Virginia Polytechnic Institute and State University, Blacksburg, VA, 24060 Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, TN 37831 3 Bredesen Center, University of Tennessee, Knoxville, TN 37996 4 Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831
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ABSTRACT Aqueous two-phase systems and related emulsion-based structures defined within micro- and nanoscale environments enable a bottom-up synthetic biological approach to mimicking the dynamic compartmentation of biomaterial that naturally occurs within cells. Model systems we have developed to aid in understanding these phenomena include on-demand generation and triggering of reversible phase transitions in ATPS confined in microscale droplets, morphological changes in networks of femtoliter-volume aqueous droplet interface bilayers (DIBs) formulated in microfluidic channels, and temperature-driven phase transitions in interfacial lipid bilayer systems supported on micro and nanostructured substrates. For each of these cases, the dynamics were intimately linked to changes in the chemical potential of water, which becomes increasingly susceptible to confinement and crowding. At these length scales, where interfacial and surface areas predominate over compartment volumes, both evaporation and osmotic forces become enhanced relative to ideal dilute solutions. Consequences of confinement and crowding in cell-sized microcompartments for increasingly complex scenarios will be discussed, from single-molecule mobility measurements with fluorescence correlation spectroscopy to spatiotemporal modulation of resource sharing in cell-free gene expression bursting. INTRODUCTION Macromolecular crowding and confinement have numerous thermodynamic and kinetic consequences for biochemical reaction dynamics and reactivity compared to dilute, homogenous environments.[1-4] Macromolecular crowding may help regulate weak DNA-protein binding interactions, for example, though both thermodynamic and kinetic mechanisms.[5] The most significant thermodynamic effects are related to excluded-volume interactions, the consequence of a reduction of configuration entropy available to reactants, which increases activity coefficients, and favors more compact conformational transition states. In addition to entropy, other nonspecific interactions such as electrostatic or hydrophobic effects modulate energetics. Dynamics are affected by changes in mobility for reacting molecules in restricted environments. Crowding will tend to decelerate diffusion-limited (fast) associations and accelerate slow (transition state activated complex) associations. Anomalous diffusion, characterized by a mean square displacement of a diffusing molecule that does not increase linearly with time, has been predicted in simulations of confined and crowded systems, yet much experimental evi
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