Publications
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(2024) Nature Communications. 15, 1, 883. Abstract
Realizing genetic circuits on single DNA molecules as self-encoded dissipative nanodevices is a major step toward miniaturization of autonomous biological systems. A circuit operating on a single DNA implies that genetically encoded proteins localize during coupled transcription-translation to DNA, but a single-molecule measurement demonstrating this has remained a challenge. Here, we use a genetically encoded fluorescent reporter system with improved temporal resolution and observe the synthesis of individual proteins tethered to a DNA molecule by transient complexes of RNA polymerase, messenger RNA, and ribosome. Against expectations in dilute cell-free conditions where equilibrium considerations favor dispersion, these nascent proteins linger long enough to regulate cascaded reactions on the same DNA. We rationally design a pulsatile genetic circuit by encoding an activator and repressor in feedback on the same DNA molecule. Driven by the local synthesis of only several proteins per hour and gene, the circuit dynamics exhibit enhanced variability between individual DNA molecules, and fluctuations with a broad power spectrum. Our results demonstrate that co-expressional localization, as a nonequilibrium process, facilitates single-DNA genetic circuits as dissipative nanodevices, with implications for nanobiotechnology applications and artificial cell design.
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(2023) Microbial Cell Factories. 22, 244. Abstract
Protein complex assembly facilitates the combination of individual protein subunits into functional entities, and thus plays a crucial role in biology and biotechnology. Recently, we developed quasi-twodimensional, silicon-based compartmental biochips that are designed to study and administer the synthesis and assembly of protein complexes. At these biochips, individual protein subunits are synthesized from locally confined high-density DNA brushes and are captured on the chip surface by molecular traps. Here, we investigate single-gene versions of our quasi-twodimensional synthesis systems and introduce the trap-binding efficiency to characterize their performance. We show by mathematical and computational modeling how a finite trap density determines the dynamics of protein-trap binding and identify three distinct regimes of the trap-binding efficiency. We systematically study how protein-trap binding is governed by the systems three key parameters, which are the synthesis rate, the diffusion constant and the trap-binding affinity of the expressed protein. In addition, we describe how spatially differential patterns of traps modulate the protein-trap binding dynamics. In this way, we extend the theoretical knowledge base for synthesis, diffusion, and binding in compartmental systems, which helps to achieve better control of directed molecular self-assembly required for the fabrication of nanomachines for synthetic biology applications or nanotechnological purposes.
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(2023) Journal of the American Chemical Society. 145, 17, p. 9729-9736 Abstract
Site-specific recombination is a cellular process for the integration, inversion, and excision of DNA segments that could be tailored for memory transactions in artificial cells. Here, we demonstrate the compartmentalization of cascaded gene expression reactions in a DNA brush, starting from the cell-free synthesis of a unidirectional recombinase that exchanges information between two DNA molecules, leading to gene expression turn-on/turn-off. We show that recombination yield in the DNA brush was responsive to gene composition, density, and orientation, with kinetics faster than in a homogeneous dilute bulk solution reaction. Recombination yield scaled with a power law greater than 1 with respect to the fraction of recombining DNA polymers in a dense brush. The exponent approached either 1 or 2, depending on the intermolecular distance in the brush and the position of the recombination site along the DNA contour length, suggesting that a restricted-reach effect between the recombination sites governs the recombination yield. We further demonstrate the ability to encode the DNA recombinase in the same DNA brush with its substrate constructs, enabling multiple spatially resolved orthogonal recombination transactions within a common reaction volume. Our results highlight the DNA brush as a favorable compartment to study DNA recombination, with unique properties for encoding autonomous memory transactions in DNA-based artificial cells.
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(2022) Cell-Free Gene Expression. Vol. 2433. p. 135-149 Abstract
Linear double-stranded DNA polymers coding for synthetic genes immobilized on a surface form a brush as a center for cell-free gene expression, with DNA density 102103 fold higher than in bulk solution reactions. A brush localizes the transcription-translation machinery in cell extracts or in cell-free reconstituted reactions from purified components, creating a concentrated source of RNA and proteins. Newly synthesized molecules can form circuits regulating gene expression in the same brush or adjacent ones. They can also assemble into functional complexes and machines such as ribosomal units, then analyzed by capture on prepatterned antibodies or by cascaded reactions. DNA brushes are arranged as a single center or multiple ones on a glass coverslip, in miniaturized compartments carved in silicon wafers, or in elastomeric microfluidic devices. Brushes create genetically programmable artificial cells with steady-state dynamics of protein synthesis. Here, we provide the basic procedure for surface patterning, DNA immobilization, capture of protein products on antibody traps and fluorescent imaging. The method of DNA brush surface patterning enables simple parallelization of cell-free gene expression reactions for high throughput studies with increased imaging sensitivity.
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(2021) ACS Synthetic Biology. 10, 3, p. 609-619 Abstract
The design of artificial cell models based on minimal surface-bound transcriptiontranslation 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 transcriptiontranslation 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.
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(2020) Nature Communications. 11, 5648. Abstract
Building autonomous artificial cells capable of homeostasis requires regulatory networks to gather information and make decisions that take time and cost energy. Decisions based on few molecules may be inaccurate but are cheap and fast. Realizing decision-making with a few molecules in artificial cells has remained a challenge. Here, we show decision-making by a bistable gene network in artificial cells with constant protein turnover. Reducing the number of gene copies from 105 to about 10 per cell revealed a transition from deterministic and slow decision-making to a fuzzy and rapid regime dominated by small-number fluctuations. Gene regulation was observed at lower DNA and protein concentrations than necessary in equilibrium, suggesting rate enhancement by co-expressional localization. The high-copy regime was characterized by a sharp transition and hysteresis, whereas the low-copy limit showed strong fluctuations, state switching, and cellular individuality across the decision-making point. Our results demonstrate information processing with low-power consumption inside artificial cells.
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(2020) Nature Nanotechnology. 15, 9, p. 783-791 Abstract
The assembly of protein machines in cells is precise, rapid, and coupled to protein synthesis with regulation in space and time. The assembly of natural and synthetic nanomachines could be similarly controlled by genetic programming outside the cell. Here, we present quasi-two-dimensional (2D) silicon compartments that enable programming of protein assembly lines by local synthesis from surface-immobilized DNA brushes. Using this platform, we studied the autonomous synthesis and assembly of a structural complex from a bacteriophage and a bacterial RNA-synthesizing machine. Local synthesis and surface capture of complexes provided high assembly yield and sensitive detection of spatially resolved assembly intermediates, with the 3D geometry of the compartment and the 2D pattern of brushes dictating the yield and mode of assembly steps. Localized synthesis of proteins in a single gene brush enhances their interactions, and displacement of their genes in separated brushes leads to step-by-step surface assembly. This methodology enables spatial regulation of protein synthesis, and deciphering, reconstruction and design of biological machine assembly lines.
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(2020) Science Advances. 6, 16, eaaz6020. Abstract
Ribosome biogenesis is an efficient and complex assembly process that has not been reconstructed outside a living cell so far, yet is the most critical step for establishing a self-replicating artificial cell. We recreated the biogenesis of Escherichia coil's small ribosomal subunit by synthesizing and capturing all its ribosomal proteins and RNA on a chip. Surface confinement provided favorable conditions for autonomous stepwise assembly of new subunits, spatially segregated from original intact ribosomes. Our real-time fluorescence measurements revealed hierarchal assembly, cooperative interactions, unstable intermediates, and specific binding to large ribosomal subunits. Using only synthetic genes, our methodology is a crucial step toward creation of a self-replicating artificial cell and a general strategy for the mechanistic investigation of diverse multicomponent macromolecular machines.
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Geometric control of bio-machine assembly by a genetic program on a chip(2019) Cell Free Systems Conference 2019. p. 18 Abstract
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(2018) ACS Synthetic Biology. 7, 8, p. 1829-1833 Abstract
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.
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(2018) Angewandte Chemie - International Edition. 57, 17, p. 4783-4786 Abstract
Lithographic patterning of DNA molecules enables spatial organization of cell-free genetic circuits under well-controlled experimental conditions. Here, we present a biocompatible, DNA-based resist termed \u201cBephore\u201d, which is based on commercially available components and can be patterned by both photo- and electron-beam lithography. The patterning mechanism is based on cleavage of a chemically modified DNA hairpin by ultraviolet light or electrons, and a subsequent strand-displacement reaction. All steps are performed in aqueous solution and do not require chemical development of the resist, which makes the lithographic process robust and biocompatible. Bephore is well suited for multistep lithographic processes, enabling the immobilization of different types of DNA molecules with micrometer precision. As an application, we demonstrate compartmentalized, on-chip gene expression from three sequentially immobilized DNA templates, leading to three spatially resolved protein-expression gradients.
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(2017) Proceedings Of The National Academy Of Sciences Of The United States Of America-Physical Sciences. 114, 44, p. 11609-11614 Abstract
Understanding how biochemical networks lead to large-scale non equilibrium 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.
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(2017) Chemical Society Reviews. 46, 18, p. 5620-5646 Abstract
We discuss the basic physics of the flow of micron-scale droplets in 2D geometry. Our focus is on the use of droplet ensembles to look into fundamental questions of non-equilibrium systems, such as the emergence of dynamic patterns and irreversibility. We review recent research in these directions, which demonstrate that 2D microfluidics is uniquely set to study complex out-of-equilibrium phenomena thanks to the simplicity of the underlying Stokes flow and the accessibility of lab-on-chip technology.
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(2016) Nature Nanotechnology. 11, 12, p. 1076-1081 Abstract
DNA can be programmed to assemble into a variety of shapes and patterns on the nanoscale and can act as a template for hybrid nanostructures such as conducting wires, protein arrays and field-effect transistors. Current DNA nanostructures are typically in the sub-micrometre range, limited by the sequence space and length of the assembled strands. Here we show that on a patterned biochip, DNA chains collapse into one-dimensional (1D) fibres that are 20nm wide and around 70μm long, each comprising approximately 35 co-aligned chains at its cross-section. Electron beam writing on a photocleavable monolayer was used to immobilize and pattern the DNA molecules, which condense into 1D bundles in the presence of spermidine. DNA condensation can propagate and split at junctions, cross gaps and create domain walls between counterpropagating fronts. This system is inherently adept at solving probabilistic problems and was used to find the possible paths through a maze and to evaluate stochastic switching circuits. This technique could be used to propagate biological or ionic signals in combination with sequence-specific DNA nanotechnology or for gene expression in cell-free DNA compartments.
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(2016) Proceedings of the National Academy of Sciences of the United States of America. 113, 29, p. 8133-8138 Abstract
Synthetic gene circuits are emerging as a versatile means to target cancer with enhanced specificity by combinatorial integration of multiple expression markers. Such circuits must also be tuned to be highly sensitive because escape of even a few cells might be detrimental. However, the error rates of decision-making circuits in light of cellular variability in gene expression have so far remained unexplored. Here, we measure the single-cell response function of a tunable logic AND gate acting on two promoters in heterogeneous cell populations. Our analysis reveals an inherent tradeoff between specificity and sensitivity that is controlled by the AND gate amplification gain and activation threshold. We implement a tumor-mimicking cellculture model of cancer cells emerging in a background of normal ones, and show that molecular parameters of the synthetic circuits control specificity and sensitivity in a killing assay. This suggests that, beyond the inherent tradeoff, synthetic circuits operating in a heterogeneous environment could be optimized to efficiently target malignant state with minimal loss of specificity.
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(2015) Nature Physics. 11, 12, p. 1037-1041 Abstract
Living systems employ front propagation and spatiotemporal patterns encoded in biochemical reactions for communication, self-organization and computation1,2,3,4. Emulating such dynamics in minimal systems is important for understanding physical principles in living cells5,6,7,8 and in vitro9,10,11,12,13,14. Here, we report a one-dimensional array of DNA compartments in a silicon chip as a coupled system of artificial cells, offering the means to implement reactiondiffusion dynamics by integrated genetic circuits and chip geometry. Using a bistable circuit we programmed a front of protein synthesis propagating in the array as a cascade of signal amplification and short-range diffusion. The front velocity is maximal at a saddle-node bifurcation from a bistable regime with travelling fronts to a monostable regime that is spatially homogeneous. Near the bifurcation the system exhibits large variability between compartments, providing a possible mechanism for population diversity. This demonstrates that on-chip integrated gene circuits are dynamical systems driving spatiotemporal patterns, cellular variability and symmetry breaking.
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(2014) Science. 345, 6198, p. 829-832 Abstract
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.
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(2014) Accounts of Chemical Research. 47, 6, p. 1912-1921 Abstract
ConspectusThe expression of genes in a cell in response to external signals or internal programs occurs within an environment that is compartmentalized and dense. Reconstituting gene expression in man-made systems is relevant for the basic understanding of gene regulation, as well as for the development of applications in bio- and nanotechnology.DNA polymer brushes assembled on a surface emulate a dense cellular environment. In a regime of significant chain overlap, the highly charged nature of DNA, its entropic degrees of freedom, and its interaction with transcription/translation machinery lead to emergent collective biophysical and biochemical properties, which are summarized in this Account.First, we describe a single-step photolithographic biochip on which biomolecules can be immobilized. Then, we present the assembly of localized DNA brushes, a few kilo-base pairs long, with spatially varying density, reaching a DNA concentration of ∼107 base pairs/μm3, which is comparable to the value in E. coli.We then summarize the response of brush height to changes in density and mono- and divalent ionic strength. The balance between entropic elasticity and swelling forces leads to a rich phase behavior. At no added salt, polymers are completely stretched due to the osmotic pressure of ions, and at high salt they assume a relaxed coil conformation. Midrange, the brush height scales with ratio of density and ionic strength to the third power, in agreement with the general theory of polyelectrolyte brushes. In response to trivalent cations, DNA brushes collapse into macroscopic dendritic condensates with hysteresis, coexistence, and a hierarchy of condensation with brush density.We next present an investigation of RNA transcription in the DNA brush. In general, the brush density entropically excludes macromolecules, depleting RNA polymerase concentration in the brush compared to the bulk, therefore reducing transcription rate. The orientation of transcription promoters with respect to the surface also affects the rate with a lower value for outward compared to inward transcription, likely due to local changes of RNA polymerase concentrations. We hypothesize that equalizing the macromolecular osmotic pressure between bulk and brush with the addition of inert macromolecules would overcome the entropic exclusion of DNA associated proteins, and lead to enhanced biochemical activity.Finally, we present protein synthesis cascades in DNA brushes patterned at close proximity, as a step toward biochemical signaling between brushes. Examining the synthesis of proteins polymerizing into crystalline tubes suggests that on-chip molecular traps serve as nucleation sites for protein assembly, thereby opening possibilities for reconstituting nanoscale protein assembly pathways.
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(2014) Journal of the American Chemical Society. 136, 13, p. 4945-4953 Abstract
We investigated the collective conformational response of DNA polymer brushes to condensation induced by the trivalent cation spermidine. DNA brushes, a few kilobase-pairs long, undergo a striking transition into macroscopic domains of collapsed chains with fractal dendritic morphology. Condensation is initiated by focal nucleation of a towerlike bundle, which laterally expands in a chain-reaction cascade of structural chain-to-chain collapse onto the surface. The transition exhibits the hallmarks of a first-order phase transition with grain boundaries, hysteresis, and coexistence between condensed and uncondensed phases. We found that an extended DNA conformation is maintained throughout the transition and is a prerequisite for the formation of large-scale dendritic domains. We identified a critical DNA density above which the nucleation propensity and growth rate sharply increase. We hypothesize that the ability of DNA-scaffolding proteins to modify the local DNA density within a genome may act as a dynamic and sensitive mechanism for spatial regulation of DNA transactions in vivo by selective condensation of chromosomal territories. By assembling a DNA brush along a patterned line narrower than twice the DNA contour length and tuning the local surface densities, we were able to initiate nucleation at a predefined location and induced growth of a single condensed nanowire over a distance 2 orders of magnitude longer than the single-chain contour. Our results demonstrate spatial control of condensation as a new tool for constructing DNA-based synthetic systems with important implications for regulation of DNA transactions on surfaces.
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(2014) Nature Physics. 10, 2, p. 140-144 Abstract
Dynamic restructuring and ordering are prevalent in driven many-body systems with long-range interactions, such as sedimenting particles, dusty plasmas, flocking animals and microfluidic droplets. Yet, understanding such collective dynamics from basic principles is challenging because these systems are not governed by global minimization principles, and because every constituent interacts with many others. Here, we report long-range orientational order of droplet velocities in disordered two-dimensional microfluidic droplet ensembles. Droplet velocities exhibit strong long-range correlation as 1/r 2, with a four-fold angular symmetry. The two-droplet correlation can be explained by representing the entire ensemble as a third droplet. The correlation amplitude is non-monotonous with density owing to excluded-volume effects. Our study puts forth a many-body problem with long-range interactions that is solvable from first principles owing to the reduced dimensionality, and introduces new experimental tools to address open problems in many-body non-equilibrium systems.
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(2013) Wiley Interdisciplinary Reviews-Nanomedicine And Nanobiotechnology. 5, 6, p. 613-628 Abstract
Large macromolecular assemblies are widespread in all cell types with diverse structural and functional roles. Whether localized to membranes, nuclei, or cytoplasm, multimeric protein-nucleic acid complexes may be viewed as sophisticated nanomachines, an inspiration to chemical design. The formation of large biological assemblies follows a complex and hierarchical self-assembly process via ordered molecular recognition events. Serving a paradigm for biological assembly, extensive past studies of T4 bacteriophage and bacterial ribosomes by many groups have been revealing distinct design strategies, yet these two very different multimeric complexes share common mechanistic motifs. An emerging biochip approach highlights two conceptual notions to promote the study of assembly pathways: cell-free expression provides coupling between synthesis and assembly; surface anchoring allows high-resolution imaging of structural intermediates and opens up opportunities for rewiring a network by defining unnatural scaffolds for synthetic design applications.
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(2013) Proceedings of the National Academy of Sciences of the United States of America. 110, 12, p. 4534-4538 Abstract
Cell-free gene expression in localized DNA brushes on a biochip has been shown to depend on gene density and orientation, suggesting that brushes form compartments with partitioned conditions. At high density, the interplay of DNA entropic elasticity, electrostatics, and excluded volume interactions leads to collective conformations that affect the function of DNA-associated proteins. Hence, measuring the collective interactions in dense DNA, free of proteins, is essential for understanding crowded cellular environments and for the design of cell-free synthetic biochips. Here, we assembled dense DNA polymer brushes on a biochip along a density gradient and directly measured the collective extension of DNA using evanescent fluorescence. DNA of 1 kbp in a brush undergoes major conformational changes, from a relaxed random coil to a stretched configuration, following a universal function of density to ionic strength ratio with scaling exponent of 1/3. DNA extends because of the swelling force induced by the osmotic pressure of ions, which are trapped in the brush to maintain local charge neutrality, in competition with the restoring force of DNA entropic elasticity. The measurements reveal in DNA crossover between regimes of osmotic, salted, mushroom, and quasineutral brush. It is surprising to note that, at physiological ionic strength, DNA density does not induce collective stretch despite significant chain overlap, which implies that excluded volume interactions in DNA are weak.
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(2012) Physics Reports-Review Section Of Physics Letters. 516, 3, p. 103-145 Abstract
We review non-equilibrium many-body phenomena in ensembles of 2D microfluidic droplets. The system comprises of continuous two-phase flow with disc-shaped droplets driven in a channel, at low Reynolds number of 10 -4-10 -3. The basic physics is that of an effective potential flow, governed by the 2DLaplace equation, with multiple, static and dynamic, boundaries of the droplets and the walls. The motion of the droplets induces dipolar flow fields, which mediate 1/r 2 hydrodynamic interaction between the droplets. Summation of these long-range 2D forces over droplet ensembles converges, in contrast to the divergence of the hydrodynamic forces in 3D. In analogy to electrostatics, the strong effect of boundaries on the equations of motion is calculated by means of image dipoles. We first consider the dynamics of droplets flowing in a 1D crystal, which exhibits unique phonon-like excitations, and a variety of nonlinear instabilities-all stemming from the hydrodynamic interactions. Narrowing the channel results in hydrodynamic screening of the dipolar interactions, which changes salient features of the phonon spectra. Shifting from a 1D ordered crystal to 2D disordered ensemble, the hydrodynamic interactions induce collective density waves and shocks, which are superposed on single-droplet randomized motion and dynamic clustering. These collective modes originate from density-velocity coupling, whose outcome is a 1D Burgers equation. The rich observational phenomenology and the tractable theory render 2D droplet ensembles a suitable table-top system for studying non-equilibrium many-body physics with long-range interactions.
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(2012) Nature Nanotechnology. 7, 6, p. 374-378 Abstract
Biologically active complexes such as ribosomes and bacteriophages are formed through the self-assembly of proteins and nucleic acids. Recapitulating these biological self-assembly processes in a cell-free environment offers a way to develop synthetic biodevices. To visualize and understand the assembly process, a platform is required that enables simultaneous synthesis, assembly and imaging at the nanoscale. Here, we show that a silicon dioxide grid, used to support samples in transmission electron microscopy, can be modified into a biochip to combine in situ protein synthesis, assembly and imaging. Light is used to pattern the biochip surface with genes that encode specific proteins, and antibody traps that bind and assemble the nascent proteins. Using transmission electron microscopy imaging we show that protein nanotubes synthesized on the biochip surface in the presence of antibody traps efficiently assembled on these traps, but pre-assembled nanotubes were not effectively captured. Moreover, synthesis of green fluorescent protein from its immobilized gene generated a gradient of captured proteins decreasing in concentration away from the gene source. This biochip could be used to create spatial patterns of proteins assembled on surfaces.
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(2012) Journal of the American Chemical Society. 134, 9, p. 3954-3956 Abstract
To study dense double-stranded DNA (dsDNA) polymer phases, we fabricated continuous density gradients of binding sites for assembly on a photochemical interface and measured both dsDNA occupancy and extension using evanescent fluorescence. Despite the abundance of available binding sites, the dsDNA density saturates after occupation of only a fraction of the available sites along the gradient. The spatial position at which the density saturates marks the onset of collective stretching of dsDNA, a direct manifestation of balancing entropic and excluded-volume interactions. The methodology presented here offers a new means to investigate dense dsDNA compartments.
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(2011) Physical review letters. 106, 4, 048104. Abstract
A complete gene expression reaction is reconstituted in a cell-free system comprising the entire endogenous transcription, translation, as well as mRNA and protein degradation machinery of E. coli. In dissecting the major reaction steps, we derive a coarse-grained enzymatic description of biosynthesis and degradation, from which ten relevant rate constants and concentrations are determined. Governed by zeroth-order degradation, protein expression follows a sharp transition from undetectable levels to constant-rate accumulation, without reaching steady state.
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Compartmentalization by directional gene expression(2010) European Cells and Materials. 20, SUPPL.3, p. 49 Abstract
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(2010) Proceedings of the National Academy of Sciences of the United States of America. 107, 7, p. 2836-2841 Abstract
The coalescence of basic biochemical reactions into compartments is a major hallmark of a living cell. Using surface-bound DNA and a transcription reaction, we investigate the conditions for boundary-free compartmentalization. The DNA self-organizes into a dense and ordered phase with coding sequences aligned at well-defined distances and orientation relative to the surface, imposing directionality on transcription. Unique to the surface in comparison to dilute homogeneous DNA solution, the reaction slows down early, is inhibited with increased DNA density, is favorable for surface-oriented promoters, and is robust against DNA condensation. We interpret these results to suggest that macromolecules (RNA polymerase and RNA), but not solutes (ions and nucleotides), are partitioned between immobilized DNA and the reservoir. Without any physical barrier, a nonequilibrium directional DNA transaction forms macromolecular gradients that define a compartment, thus offering a boundary-free approach to the assembly of a synthetic cell.
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(2010) Molecular Systems Biology. 6, 444. Abstract
Precise discrimination between similar cellular states is essential for autonomous decision-making scenarios, such as in vivo targeting of diseased cells. Discrimination could be achieved by delivering an effector gene expressed under a highly active context-specific promoter. Yet, a single-promoter approach has linear response and offers limited control of specificity and efficacy. Here, we constructed a dual-promoter integrator, which expresses an effector gene only when the combined activity of two internal input promoters is high. A tunable response provides flexibility in choosing promoter inputs and effector gene output. Experiments using one premalignant and four cancer cell lines, over a wide range of promoter activities, revealed a digital-like response of input amplification following a sharp activation threshold. The response function is cell dependent with its overall magnitude increasing with degree of malignancy. The tunable digital-like response provides robustness, acts to remove input noise minimizing false-positive identification of cell states, and improves targeting precision and efficacy.
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(2009) Nano Letters. 9, 12, p. 4462-4466 Abstract
We assemble on a chip spatially resolved DNA polymer brushes with density that can be controlled along continuous gradients, from dilute to dense packing, with 20-30 nm between DNA molecules. Investigation of DNA digestion in a - 1 kb DNA brush showed that endonucleases can digest within the brush, yet their activity is impeded at high DNA density and for restriction sites near the bottom. Local gene activation-a form of switch-was then demonstrated by a digestion-ligation cascade between two spatially resolved DNA brushes, leading to the in situ expression of green fluorescent protein upon a diffusion-controlled DNA swapping.
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(2009) Physical review letters. 103, 11, 114502. Abstract
We investigate the collective motion of a two-dimensional disordered ensemble of droplets in a microfluidic channel far from equilibrium and at Reynolds number □10-4. The ensemble carries ultraslow shock waves and sound, propagating at □100μms̄1 and superposed on diffusive droplets motion. These modes are induced by long-range hydrodynamic dipolar interactions between droplets, the result of the symmetry breaking flow. The modes obey the Burgers equation due to a local coupling between droplets velocity and number density. This stems from a singular effect of the channel sidewall boundaries upon summation of the hydrodynamic interaction in two dimensions.
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(2009) Small. 5, 15, p. 1723-1726 Abstract
A biochip model of lymphocyte locomotion on confined chemokine tracks is described. Micropatterned chemokine biochips were constructed with the help microscope glass coverslips coated with daisy molecular films. Chemically reactive primary amines were fully de-protected at the solution termini of daisy by shining UV light. Biochips were assembled on slides mounted on an automated stage of an inverted microscope. The slides were assembled within a hermetically sealed chamber apparatus 0.3 mm in height to avoid fluid convection. Human T lymphocytes freshly isolated from blood were perfused through the chamber. Upon entry to the chamber flow was stopped, allowing cells to randomly settle on the CXCL12-presenting biochip. Movies were recorded over 30-min periods in multiple fields of view using Softmorx 3.5 at four frames per minute using a 20x/0.95 NA differential interference contrast objective.
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(2009) Nano Letters. 9, 2, p. 909-913 Abstract
Dense brushes of linear DNA polymers are assembled on a blochlp with ∼30 nm between anchorage points, amounting to a few mega-base-pairs/ μm3. In bulk solution, a barrier Incurs to conjugate more than two end-functionalized DNAs. However, such doublets bind the surface with almost equal efficiency to singlets, suggesting that extended brush buildup reduces the barrier. On-chip transcription reveals that doublets are roughly 2-fold Inefficient compared to singlets, a manifestation of the Interaction of the enzymatic machinery with the dense brush. Synthetic gene brushes made of DNA conjugates provide simple means to regulate expression on a chip.
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(2008) Molecular Systems Biology. 4, 181. Abstract
We present the assembly of gene brushes by means of a photolithographic approach that allows us to control the density of end-immobilized linear double-stranded DNA polymers coding for entire genes. For 2 kbp DNAs, the mean distance varies from 300 nm, where DNAs are dilute and assume relaxed conformations, down to 30 nm, where steric repulsion at dense packing forces stretching out. We investigated the gene-to-protein relationship of firefly luciferase under the T7/E.Coli-extract expression system, as well as transcription-only reactions with T7 RNA polymerase, and found both systems to be highly sensitive to brush density, conformation, and orientation. A 'structure-function' picture emerges in which extension of genes induced by moderate packing exposes coding sequences and improves their interaction with the transcription/translation machinery. However, tighter packing impairs the penetration of the machinery into the brush. The response of expression to two-dimensional gene crowding at the nanoscale identifies gene brushes as basic controllable units en route to multicomponent synthetic systems. In turn, these brushes could deepen our understanding of biochemical reactions taking place under confinement and molecular crowding in living cells.
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One-dimensional microfluidic crystals far from equilibrium acoustic phonons, instabilities and confinement(2008) Progress of Theoretical Physics. 175 SUPPLEMENT, p. 123-130 Abstract
We investigated the collective motion of a one-dimensional array of water-in-oil droplets flowing in microfluidic channel in quasi-2D at low Reynolds number. Driven far from equilibrium by the symmetry-breaking flow field, the droplets exhibit acoustic normal modes (crystal 'phonons') with unusual dispersion relations. These phonons are due to long-range hydrodynamic dipolar interactions between the droplets. The phonon spectra change anomalously at the crossover between unconfined 2D flow and ID confined flow, as a result from an interplay between boundary-induced screening and crystal incompressibility. Microfluidic crystals offer a vista, in the linear flow regime, into soft-matter systems far from equilibrium.
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(2007) Science. 318, 5853, p. 1078-1079 Abstract
An amplification mechanism brings DNA circuits closer to practical applications.
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(2007) Physical Biology. 4, 3, p. 154-163 Abstract
We present an approach for an autonomous system that detects a particular state of interest in a living cell and can govern cell fate accordingly. Cell states could be better identified by the expression pattern of several genes than of a single one. Therefore, autonomous identification can be achieved by a system that measures the expression of these several genes and integrates their activities into a single output. We have constructed a system that diagnoses a unique state in yeast, in which two independent pathways, methionine anabolism and galactose catabolism, are active. Our design is based on modifications of the yeast two-hybrid system. We show that cells could autonomously report on their state, identify the state of interest, and inhibit their growth accordingly. The system's sensitivity is adjustable to detect states with limited dynamic range of inputs. The system's output depends only on the activity of input pathways, not on their identity; hence it is straightforward to diagnose any pair of inputs. A simple model is presented that accounts for the data and provides predictive power. We propose that such systems could handle real-life states-of-interest such as identification of aberrant versus normal growth.
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(2007) Physical review letters. 99, 12, 124502. Abstract
We investigate the acoustic normal modes ("phonons") of a 1D microfluidic droplet crystal at the crossover between 2D flow and confined 1D plug flow. The unusual phonon spectra of the crystal, which arise from long-range hydrodynamic interactions, change anomalously under confinement. The boundaries induce weakening and screening of the interactions, but when approaching the 1D limit we measure a marked increase in the crystal sound velocity, a sign of interaction strengthening. This nonmonotonous behavior of the phonon spectra is explained theoretically by the interplay of screening and plug flow.
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(2007) Small. 3, 3, p. 500-510 Abstract
We have developed a biochip platform technology suitable for controlled cell-free gene expression at the micrometer scale. A new hybrid molecule, "Daisy", was designed and synthesized to form in a single step a biocompatible lithographic interface on silicon dioxide. A protocol is described for the immobilization of linear DNA molecules thousands of base pairs long on Daisy-coated surfaces with submicrometer spatial resolution and up to high densities. On-chip protein synthesis can be obtained with a dynamic range of up to four orders of magnitude and minimal nonspecific activity. En route to on-chip artificial gene circuits, a simple two-stage gene cascade was built, in which the protein synthesized at the first location diffuses to regulate the synthesis of another protein at a second location. We demonstrate the capture of proteins from crude extract onto micrometer-scale designated traps, an important step for the formation of miniaturized self-assembled protein chips. Our biochip platform can be combined with elastomeric microfluidic devices, thereby opening possibilities for isolated and confined reaction chambers and artificial cells in which the transport of products and reagents is done by diffusion and flow. The Daisy molecule and described approach enables groups not proficient in surface chemistry to construct active biochips based on cellfree gene expression.
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(2007) Nano Letters. 7, 3, p. 638-641 Abstract
We used a cell-free transcription/translation system to synthesize structural proteins of the T4 bacteriophage. We focused on two proteins that participate in the formation of the virus tail tube assembly. Synthesized separately, the proteins assembled into their in vivo forms, namely one polymerized into rigid hollow nanotubes 20 nm thick and hundreds of nanometers long, the other assembled into 10 nm tube-capping hexameric rings. Co-synthesis of the two proteins, however, revealed a novel structure of a nanodoughnut with an outer diameter of 50 nm and thickness of ∼20 nm. Cell-free co-synthesis and assembly of T4 structural proteins can be extended in a combinatorial fashion. The addition of other structural genes offers control of native nanoassemblies and may reveal ones not observable by mixing purified components.
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(2006) Nature Physics. 2, 11, p. 743-748 Abstract
The development of a general theoretical framework for describing the behaviour of a crystal driven far from equilibrium has proved difficult. Microfluidic crystals, formed by the introduction of droplets of immiscible fluid into a liquid-filled channel, provide a convenient means to explore and develop models to describe non-equilibrium dynamics. Owing to the fact that these systems operate at low Reynolds number (Re), in which viscous dissipation of energy dominates inertial effects, vibrations are expected to be over-damped and contribute little to their dynamics. Against such expectations, we report the emergence of collective normal vibrational modes (equivalent to acoustic 'phonons') in a one-dimensional microfluidic crystal of water-in-oil droplets at Re ∼ 10 -4 . These phonons propagate at an ultra-low sound velocity of ∼ 100 μm s -1 and frequencies of a few hertz, exhibit unusual dispersion relations markedly different to those of harmonic crystals, and give rise to a variety of crystal instabilities that could have implications for the design of commercial microfluidic systems. First-principles theory shows that these phonons are an outcome of the symmetry-breaking flow field that induces long-range inter-droplet interactions, similar in nature to those observed in many other systems including dusty plasma crystals, vortices in superconductors, active membranes and nucleoprotein filaments.
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(2006) Protein Expression and Purification. 48, 2, p. 243-252 Abstract
The Tenebrio molitor thermal hysteresis protein has a cysteine content of 19%. This 84-residue protein folds as a compact β-helix, with eight disulfide bonds buried in its core. Exposed on one face of the protein is an array of threonine residues, which constitutes the ice-binding face. Previous protocols for expression of this protein in recombinant expression systems resulted in inclusion bodies or soluble but largely inactive material. A long and laborious refolding procedure was performed to increase the fraction of active protein and isolate it from inactive fractions. We present a new protocol for production of fully folded and active T. molitor thermal hysteresis protein in bacteria, without the need for in vitro refolding. The protein coding sequence was fused to those of various carrier proteins and expressed at low temperature in a bacterial strain specially suited for production of disulfide-bonded proteins. The product, after a simple and robust purification procedure, was analyzed spectroscopically and functionally and was found to compare favorably to previously published data on refolded protein and protein obtained from its native source.
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(2005) Physical Biology. 2, 3, p. P1-P8 Abstract
We present a new experimental approach to build an artificial cell using the translation machinery of a cell-free expression system as the hardware and a DNA synthetic genome as the software. This approach, inspired by the self-replicating automata of von Neumann, uses cytoplasmic extracts, encapsulated in phospholipid vesicles, to assemble custom-made genetic circuits to develop the functions of a minimal cell. Although this approach can find applications, especially in biotechnology, the primary goal is to understand how a DNA algorithm can be designed to build an operating system that has some of the properties of life. We provide insights on this cell-free approach as well as new results to transform step by step a long-lived vesicle bioreactor into an artificial cell. We show how the green fluorescent protein can be anchored to the membrane and we give indications of a possible insertion mechanism of integral membrane proteins. With vesicles composed of different phospholipids, the fusion protein alpha-hemolysin-eGFP can be expressed to reveal patterns on the membrane. The specific degradation complex ClpXP from E. coli is introduced to create a sink for the synthesized proteins. Perspectives and subsequent limitations of this approach are discussed.
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(2004) Physical review letters. 93, 25, 258103. Abstract
The binding fluctuations of protein, RecA, were used to analyze the high-fidelity DNA sensing. Fluorescence anisotropy measurements at the binding onset were used to show that adenosine triphosphate (ATP) consumption enhanced DNA length discrimination. A model was developed which describes the DNA length sensing as an outcome of out-of-equilibrium binding fluctuations. It was observed that the cascade architecture of the binding fluctuations was a generalization of the kinetic proofreading mechanism.
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(2003) Proceedings of the National Academy of Sciences of the United States of America. 100, 22, p. 12672-12677 Abstract
Cell-free genetic circuit elements were constructed in a transcription-translation extract. We engineered transcriptional activation and repression cascades, in which the protein product of each stage is the input required to drive or block the following stage. Although we can find regions of linear response for single stages, cascading to subsequent stages requires working in nonlinear regimes. Substantial time delays and dramatic decreases in output production are incurred with each additional stage because of a bottleneck at the translation machinery. Faster turnover of RNA message can relieve competition between genes and stabilize output against variations in input and parameters.
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(2002) Proceedings of the National Academy of Sciences of the United States of America. 99, 18, p. 11589-11592 Abstract
The assembly of RecA on single-stranded DNA is measured and interpreted as a stochastic finite-state machine that is able to discriminate fine differences between sequences, a basic computational operation. RecA filaments efficiently scan DNA sequence through a cascade of random nucleation and disassembly events that is mechanistically similar to the dynamic instability of microtubules. This iterative cascade is a multistage kinetic proofreading process that amplifies minute differences, even a single base change. Our measurements suggest that this stochastic Turing-like machine can compute certain integral transforms.
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(2001) Proceedings of the National Academy of Sciences of the United States of America. 98, 16, p. 9068-9073 Abstract
Fluorescence anisotropy is used to follow the binding of RecA to short single-stranded DNA (ssDNA) sequences (39 bases) at low DNA and RecA concentration where the initial phase of polymerization occurs. We observe that RecA condensation is extremely sensitive to minute changes in DNA sequences. RecA binds strongly to sequences that are rich in pyrimidines and that lack significant secondary structure and base stacking. We find a correlation between the DNA folding free energy and the onset concentration for RecA binding. These results suggest that the folding of ssDNA and base stacking represent a barrier for RecA binding. The link between secondary structure and binding affinity is further analyzed with two examples: discrimination between two naturally occurring polymorphisms differing by one base and RecA binding on a molecular beacon. A self-assembly model is introduced to explain these observations. We propose that RecA may be used to sense ssDNA sequence and structure.
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(1999) Proceedings of the National Academy of Sciences of the United States of America. 96, 18, p. 10140-10145 Abstract
Gradual disruption of the actin cytoskeleton induces a series of structural shape changes in cells leading to a transformation of cylindrical cell extensions into a periodic chain of 'pearls.' Quantitative measurements of the pearling instability give a square-root behavior for the wavelength as a function of drug concentration. We present a theory that explains these observations in terms of the interplay between rigidity of the submembranous actin shell and tension that is induced by boundary conditions set by adhesion points. The theory allows estimation of the rigidity and thickness of this supporting shell. The same theoretical considerations explain the shape of nonadherent edges in the general case of untreated cells.
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(1998) Biophysical Journal. 75, 1, p. 294-320 Abstract
We present the phenomenology of transformations in lipid bilayers that are excited by laser tweezers. A variety of dynamic instabilities and shape transformations are observed, including the pearling instability, expulsion of vesicles, and more exotic ones, such as the formation of passages. Our physical picture of the laser-membrane interaction is based on the generation of tension in the bilayer and loss of surface area. Although tension is the origin of the pearling instability, it does not suffice to explain expulsion of vesicles, where we observe opening of giant pores and creeping motion of bilayers. We present a quantitative theoretical framework to understand most of the observed phenomenology. The main hypothesis is that lipid is pulled into the optical trap by the familiar dielectric effect, is disrupted, and finally is repackaged into an optically unresolvable suspension of colloidal particles. This suspension, in turn, can produce osmotic pressure and depletion forces, driving the observed transformations.
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(1998) Biophysical Journal. 74, 3, p. 1541-1548 Abstract
We present a new approach to probing single-particle dynamics that uses dynamic light scattering from a localized region. By scattering a focused laser beam from a micron-size particle, we measure its spatial fluctuations via the temporal autocorrelation of the scattered intensity. We demonstrate the applicability of this approach by measuring the three-dimensional force constants of a single bead and a pair of beads trapped by laser tweezers. The scattering equations that relate the scattered intensity autocorrelation to the particle position correlation function are derived. This technique has potential applications for measurement of biomolecular force constants and probing viscoelastic properties of complex media.
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(1998) Physical review letters. 81, 8, p. 1738-1741 Abstract
We present a new approach for determining optical gradient forces applied by strongly focused laser beams on dielectric particles. We show that when the electromagnetic field is focused to a diffraction limited spot a dipole approximation is valid for any particle size. We derive intuitive predictions for force-displacement curves, maximal trapping forces, and force constants. The theory fits well with recent measurements of particles trapped by laser tweezers. We also discuss effects of radiation pressure and gravity.
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(1997) Physical review letters. 78, 2, p. 386-389 Abstract
Irradiation of a giant unilamellar lipid bilayer vesicle with a focused laser spot leads to a tense pressurized state which persists indefinitely after laser shutoff. If the vesicle contains another object it can then be gently and continuously expelled from the tense outer vesicle. Remarkably, the inner object can be almost as large as the parent vesicle; its volume is replaced during the exit process. We offer a qualitative theoretical model to explain these and related phenomena. The main hypothesis is that the laser trap pulls in lipid and ejects it in the form of submicron objects, whose osmotic activity then drives the expulsion.
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(1997) Physical review letters. 78, 1, p. 154-157 Abstract
We developed an experimental technique which probes the dynamics of a single colloidal particle over many decades in time, with spatial resolution of a few nanometers. By scattering a focused laser beam from a particle observed in an optical microscope, we measure its fluctuations via the temporal autocorrelation function of the scattered intensity g\(t\). This technique is demonstrated by applying it to a single Brownian particle in an optical trap of force constant k. The decay times of g\(t\), which are related to the particle position autocorrelation function, scale as k−1, as expected from theory.
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(1997) Physical review letters. 79, 6, p. 1158-1161 Abstract
We report quantitative measurements above threshold in the pearling instability of cylindrical membranes. Induced by optical tweezers, the instability propagates outward from the laser trap with well defined wavelength, and velocity. All measured quantities scale with the reduced tension control parameter ε ≡ (Σ - Σc)/Σc. A critical slowing down for initiation of the instability is observed. The values of the velocity, wavelength, and delay time agree with marginal stability and linear analysis. Measurements very close to threshold are strongly masked by thermal fluctuations.
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(1995) Physical review letters. 75, 18, p. 3356-3359 Abstract
Loosely bound membranes exhibit an unusual elastic response when pinched together by optical tweezers, locally unbinding to a large intermembrane distance. Tweezing a stack of many bound membranes produces extreme local swelling in the vicinity of the tweezing point. We introduce a model that incorporates bending elasticity, fluctuations, and intermembrane interactions to calculate the membrane profiles subject to a local pinch. Theoretically, we find strongly overshooting profiles in agreement with experiment. We predict scaling behavior of the overshoot with the pinch strength and size.
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(1995) Physical review letters. 75, 19, p. 3481-3484 Abstract
We report experimental observations and theoretical analysis of entropy driven expulsion in giant lipid vesicles, produced by the application of optical tweezers. The tweezers induce tension in membranes by attracting lipid material into the optical trap. This suppresses fluctuations in the vesicles and converts them into tense pressurized spheres. After shutting off the laser, overpressurized vesicles spontaneously expel inner vesicles, releasing their inner pressure.
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(1994) Physical review letters. 73, 10, p. 1392-1395 Abstract
We investigate the stability of tubular fluid membranes by perturbing them with optical tweezers. A peristaltic instability appears, with wavelength on the order of the tube circumference, characterized by tautness and suppression of curvature fluctuations in the membrane. We interpret this in terms of a model that includes a surface tension term in the elastic energy, and describes a transition to stable, finite amplitude peristaltic states. At high amplitudes the experiment reveals new dynamic states of "pearls" interconnected via thin tubes along which they travel and aggregate.
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(1994) Physical Review E. 49, 5, p. 4306-4321 Abstract
Recent experiments that probe the effect of alcohol monolayers on the freezing of water are an example of well-characterized surface nucleation, where one has control over the instability by systematic surface modification. We present a simple theory of surface-modified, first-order phase transitions and show how supercooling may in fact be inhibited below a minimal supercooling temperature which is dependent on the macroscopic strength and spatial extent of the surface treatment. The results show that the temperature range where supercooling is possible can indeed vanish for strong enough surface treatments, in qualitative agreement with the experiments.
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(1993) Langmuir. 9, 11, p. 2786-2788 Abstract
We apply the continuum theory of van der Waals interactions to a four-component, layered system, composed of a bulk vapor phase, a thin hydrocarbon film, and a water film which is in equilibrium with bulk ice, at the triple point. We find that for thin hydrocarbon films, these interactions result in a finite film of water at the ice surface with a thickness which increases with the hydrocarbon film thickness, d, reaching a value of about 1000 Å for d ≈ 300 Å. This corresponds to incomplete surface melting of ice but with a relatively thick wetting layer. However, for larger d, we predict a discontinuous wetting transition as the water film thickness jumps to infinity, indicating complete surface melting of ice.