[Show abstract][Hide abstract] ABSTRACT: Cell-free systems offer a simplified and flexible context that enables important biological reactions while removing complicating factors such as fitness, division, and mutation that are associated with living cells. However, cell-free expression in unconfined spaces is missing important elements of expression in living cells. In particular, the small volume of living cells can give rise to significant stochastic effects, which are negligible in bulk cell-free reactions. Here, we confine cell-free gene expression reactions to cell-relevant 20 fL volumes (between the volumes of E. coli and S. cerevisiae), in polydimethylsiloxane (PDMS) containers. We demonstrate that expression efficiency varies widely among different containers, likely due to non-Poisson distribution of expression machinery at the observed scale. Previously, this phenomenon has been observed only in liposomes. In addition, we analyze gene expression noise. This analysis is facilitated by our use of cell-free systems, which allow the mapping of the measured noise properties to intrinsic noise models. In contrast, previous live cell noise analysis efforts have been complicated by multiple noise sources. Noise analysis reveals signatures of translational bursting while noise dynamics suggest that overall cell-free expression is limited by a diminishing translation rate. In addition to offering a unique approach to understanding noise in gene circuits, our work contributes to a deeper understanding of the biophysical properties of cell-free expression systems, thus aiding efforts to harness cell-free systems for synthetic biology applications.
[Show abstract][Hide abstract] ABSTRACT: We present neutron reflectivity measurements of the in-situ microscopic
architecture of phospholipid molecules at the interface between a
regularly nano-patterned surface and an aqueous sub-phase using neutron
reflectometry. 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) single
bilayers were deposited on a patterned silicon substrate. The substrate
was patterned with a rectangular array of nano-scaled holes using e-beam
nano-lithographic techniques. The goal of these experiments is to
produce a set of small freely-suspended bilayers spanning the
nanostructured surface. We compare results for films deposited by
vesicle adsorption or by the Langmuir--Shafer (L-S) technique. Initial
data analysis shows that there are well formed bilayers on the surface.
Detailed analysis of the reflectivity curves will be presented to
confirm details of the architecture of these bilayer films. Bilayers
prepared in this way may serve as model single bilayer systems with
freely suspended areas for the study of membrane functionality in
biological and biomimetic materials and systems.
[Show abstract][Hide abstract] ABSTRACT: Droplet-based microfluidic platforms offer many opportunities to confine chemical and biochemical reactants in discrete ultrasmall reaction volumes, and investigate the effects of increased confinement on reaction dynamics. Current state-of-the-art microfluidic sampling strategies for creating ultrasmall reaction volumes are predominately steady-state approaches, which result in difficulty in trapping reacting species with a well-defined time-zero for initiation of biochemical reactions in the confined space. This talk describes stepwise, on-demand generation and fusion of femtoliter aqueous droplets based on interfacial tension. Sub-millisecond reaction times from droplet fusion were demonstrated, as well as a reversible chemical toggle switch based on alternating fusion of droplets containing acidic or basic solution, monitored with the pH-dependent emission of fluorescein.
[Show abstract][Hide abstract] ABSTRACT: We describe a method for creating discrete femtolitre-scale water-in-oil droplets on demand, based solely on a geometrically induced reduction in oil/water interfacial area at microfabricated junction orifices. This on-demand generation method is driven by self-shear of droplets due to interfacial tension induced forces resulting from a localized transition in microchannel height. The magnitudes of shear stresses involved appear to be significantly less than the shearing instabilities used to split off daughter droplets from aqueous mother plugs at microfabricated junctions in continuous water-in-oil segmented flows, which implies that this method may be better suited for studying biochemical reactions and reaction kinetics in droplets of decreased volume without loss of chemical reactivity due to redistribution of surfactant density used to passivate the oil/water interface. Predictable droplet generation rates under constant pressure conditions or the gated formation of one, two or more droplets at a time with fixed pressure pulses have been demonstrated in a similar manner to active on-demand droplet generation strategies, but with a simpler system not needing actuation and sensing equipment beyond a pressure regulator.
Lab on a Chip 10/2010; 10(20):2688-94. · 5.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This paper describes stepwise on-demand generation and fusion of femtolitre aqueous droplets based on interfacial tension. Sub-millisecond reaction times from droplet fusion were demonstrated, as well as a reversible chemical toggle switch based on alternating fusion of droplets containing acidic or basic solution, monitored with the pH-dependent emission of fluorescein.
Lab on a Chip 10/2010; 10(24):3373-6. · 5.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We developed a microfluidic platform for splitting well-mixed, femtoliter-volume droplets from larger water-in-oil plugs, where the sizes of the daughter droplets were not limited by channel width. These droplets were separated from mother plugs at a microfabricated T-junction, which enabled the study of how increased confinement affected enzyme kinetics in droplets 4-10 microm in diameter. Initial rates for enzyme catalysis in the mother plugs and the largest daughter drops were close to the average bulk rate, while the rates in smaller droplets decreased linearly with increasing surface to volume ratio. Rates in the smallest droplets decreased by a factor of 4 compared to the bulk rate. Traditional methods for detecting nonspecific adsorption at the water-oil interface were unable to detect evidence of enzyme adsorption, including pendant drop tensiometry, laser scanning confocal microscopy of drops containing labeled proteins in microemulsions, and epifluorescence microscopy of plugs and drops generated on-chip. We propose the slowing of enzyme reaction kinetics in the smaller droplets was the result of increased adsorption and inactivation of enzymes at the water-oil interface arising from transient interfacial shear stresses imparted on the daughter droplets as they split from the mother plugs and passed through the constricted opening of the T-junction. Such stresses are known to modulate the interfacial area and density of surfactant molecules that can passivate the interface. Bright field images of the splitting processes at the junction indicate that these stresses scaled with increasing surface to volume ratios of the droplets but were relatively insensitive to the average flow rate of plugs upstream of the junction.
[Show abstract][Hide abstract] ABSTRACT: A device with femtoliter-scale chambers and controlled reaction initiation was developed for single-molecule enzymology. Initially separated substrate and enzyme streams were rapidly mixed in a microfluidic device and encapsulated in an array of individual microreactors, allowing for enzyme kinetics to be monitored with millisecond dead times and single-molecule sensitivity. Because the arrays of chambers were fabricated by micromolding in PDMS, the chambers were monodisperse in size, and the chamber volume could be systematically controlled. Microreactors could be purged and replenished with fresh reactants for consecutive rounds of observation. Repeated experiments with statistically identical initial conditions could be performed rapidly, with zero cross-talk between chambers in the array.
[Show abstract][Hide abstract] ABSTRACT: Since their initial fabrication two decades ago by McConnell and coworkers, fluid supported phospholipid bilayers (SLBs) have
played a key role in the development of nanoscale assemblies of biological materials on artificial supports.1,2 The reason for this is quite straightforward. SLBs can serve as biomimetics for chemical and biological processes which occur
in cell membranes. A thin aqueous layer (approximately 1 nm thick) is trapped between the bilayer and the underlying support
(Figure 6.1). Thiswater layer acts as a lubricant allowing both leaflets of the bilayer to remain fluid.3–10 Consequently, planar supported membranes retain many of the physical properties of free vesicles or even native cell surfaces
when the appropriate recognition components are present.4 Specifically, SLBs are capable of undergoing lateral rearrangements to accommodate binding by aqueous proteins, viruses,
toxins, and even cells.11 As substrate supported entities, they are convenient to study by a host of interface-sensitive techniques and are far less
fragile than either unsupported membranes or full-blown cellular systems.
[Show abstract][Hide abstract] ABSTRACT: Solid supported lipid bilayers are rapidly delaminated when drawn through the air/water interface. We have discovered that a close packed monolayer of specifically bound protein prevents this process. The protection mechanism worked in two ways. First, when protein-protected bilayers were drawn through the air/water interface, a thin bulk water layer was visible over the entire bilayer region, thereby preventing air from contacting the surface. Second, a stream of nitrogen was used to remove all bulk water from a protected bilayer, which remained fully intact as determined by fluorescence microscopy. The condition of this dried bilayer was further probed by fluorescence recovery after photobleaching. It was found that lipids were not two-dimensionally mobile in dry air. However, when the bilayer was placed in a humid environment, 91% of the bleached fluorescence signal was recovered, indicating long-range two-dimensional mobility. The diffusion coefficient of lipids under humid conditions was an order of magnitude slower than the same bilayer under water. Protected bilayers could be rehydrated after drying, and their characteristic diffusion coefficient was reestablished. Insights into the mechanism of bilayer preservation were suggested.
Journal of the American Chemical Society 07/2004; 126(21):6512-3. · 10.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have developed a general method for photopatterning well-defined patches of enzymes inside a microfluidic device at any location. First, a passivating protein layer was adsorbed to the walls and floor of a poly(dimethylsiloxane)/glass microchannel. The channel was then filled with an aqueous biotin-linked dye solution. Using an Ar+/Kr+ laser, the fluorophore moieties were bleached to create highly reactive species. These activated molecules subsequently attached themselves to the adsorbed proteins on the microchannel walls and floor via a singlet oxygen-dependent mechanism. Enzymes linked to streptavidin or avidin could then be immobilized via (strept)avidin/biotin binding. Using this process, we were able to pattern multiple patches of streptavidin-linked alkaline phosphatase inside a straight microfluidic channel without the use of valves under exclusively aqueous conditions. The density of alkaline phosphatase in the patches was calculated to be approximately 5% of the maximum possible density by comparison with known standards. Turnover was observed via fluorogenic substrate conversion and fluorescence microscopy. A more complex two-step enzyme reaction was also designed. In this case, avidin-linked glucose oxidase and streptavidin-linked horseradish peroxidase were sequentially patterned in separate patches inside straight microfluidic channels. Product formed at the glucose oxidase patch became the substrate for horseradish peroxidase, patterned downstream, where fluorogenic substrate turnover was recorded.
[Show abstract][Hide abstract] ABSTRACT: The molecular level details of the displacement of surface adsorbed fibrinogen from silica substrates were studied by atomic force microscopy, immunochemical assays, fluorescence microscopy, and vibrational sum frequency spectroscopy. The results showed that human plasma fibrinogen (HPF) can be readily displaced from the interface by other plasma proteins near neutral pH because the positively charged alpha C domains on HPF sit between the rest of the macromolecule and the underlying surface. The alpha C domains make weak electrostatic contact with the substrate, which is manifest by a high degree of alignment of Lys and Arg residues. Upon cycling through acidic pH, however, the alpha C domains are irreversibly removed from this position and the rest of the macromolecule is free to engage in stronger hydrogen bonding, van der Waals, and hydrophobic interactions with the surface. This results in a 170-fold decrease in the rate at which HPF can be displaced from the interface by other proteins in human plasma.
Journal of the American Chemical Society 11/2003; 125(42):12782-6. · 10.68 Impact Factor