Publikationen

Zusammenfassung

The generation of a chemical system capable of replication and evolution is a key objective of synthetic biology. This could be achieved by in vitro reconstitution of a minimal self-sustaining central dogma consisting of DNA replication, transcription and translation. Here, we present an in vitro translation system, which enables self-encoded replication and expression of large DNA genomes under well-defined, cell-free conditions. In particular, we demonstrate self-replication of a multipartite genome of more than 116 kb encompassing the full set of Escherichia coli translation factors, all three ribosomal RNAs, an energy regeneration system, as well as RNA and DNA polymerases. Parallel to DNA replication, our system enables synthesis of at least 30 encoded translation factors, half of which are expressed in amounts equal to or greater than their respective input levels. Our optimized cell-free expression platform could provide a chassis for the generation of a partially self-replicating in vitro translation system.

Zusammenfassung

To advance microfluidic integration, we present the use of two-photon additive manufacturing to fold 2D channel layouts into compact free-form 3D fluidic circuits with nanometer precision. We demonstrate this technique by tailoring microfluidic nozzles and mixers for time-resolved structural biology at X-ray free-electron lasers (XFELs). We achieve submicron jets with speeds exceeding 160 m s−1, which allows for the use of megahertz XFEL repetition rates. By integrating an additional orifice, we implement a low consumption flow-focusing nozzle, which is validated by solving a hemoglobin structure. Also, aberration-free in operando X-ray microtomography is introduced to study efficient equivolumetric millisecond mixing in channels with 3D features integrated into the nozzle. Such devices can be printed in minutes by locally adjusting print resolution during fabrication. This technology has the potential to permit ultracompact devices and performance improvements through 3D flow optimization in all fields of microfluidic engineering.

Zusammenfassung

Point-of-care testing (POCT) in low-resource settings requires tools that can operate independently of typical laboratory infrastructure. Due to its favorable signal-to-background ratio, a wide variety of biomedical tests utilize fluorescence as a readout. However, fluorescence techniques often require expensive or complex instrumentation and can be difficult to adapt for POCT. To address this issue, we developed a pocket-sized fluorescence detector costing less than $15 that is easy to manufacture and can operate in low-resource settings. It is built from standard electronic components, including an LED and a light dependent resistor, filter foils and 3D printed parts, and reliably reaches a lower limit of detection (LOD) of ≈ 6.8 nM fluorescein, which is sufficient to follow typical biochemical reactions used in POCT applications. All assays are conducted on filter paper, which allows for a flat detector architecture to improve signal collection. We validate the device by quantifying in vitro RNA transcription and also demonstrate sequence-specific detection of target RNAs with an LOD of 3.7 nM using a Cas13a-based fluorescence assay. Cas13a is an RNA-guided, RNA-targeting CRISPR effector with promiscuous RNase activity upon recognition of its RNA target. Cas13a sensing is highly specific and adaptable and in combination with our detector represents a promising approach for nucleic acid POCT. Furthermore, our open-source device may be used in educational settings, through providing low cost instrumentation for quantitative assays or as a platform to integrate hardware, software and biochemistry concepts in the future.

Zusammenfassung

A biomimetic system capable of replication and segregation of genetic material constitutes an essential component for the future design of a minimal synthetic cell. Here we have used the simple T7 bacteriophage system and the plasmid‐derived ParMRC system to establish in vitro DNA replication and DNA segregation, respectively. These processes were incorporated into biomimetic compartments providing an enclosed reaction space. The functional lifetime of the encapsulated segregation system could be prolonged by equipping it with ATP‐regenerating and oxygen‐scavenging systems. Finally, we showed that DNA replication and segregation processes could be coupled in vitro by using condensed DNA nanoparticles resulting from DNA replication. ParM spindles extended over tens of micrometers and could thus be used for segregation in compartments that are significantly longer than bacterial cell size. Overall, this work demonstrates the successful bottom‐up assembly and coupling of molecular machines that mediate replication and segregation, thus providing an important step towards the development of a fully functional minimal cell.

Zusammenfassung

The new European X-ray Free-Electron Laser (European XFEL) is the first X-ray free-electron laser capable of delivering intense X-ray pulses with a megahertz interpulse spacing in a wavelength rang...

Zusammenfassung

RNA replicases catalyse transcription and replication of viral RNA genomes. Of particular interest for in vitro studies are phage replicases due to their small number of host factors required for activity and their ability to initiate replication in the absence of any primers. However, the requirements for template recognition by most phage replicases are still only poorly understood. Here, we show that the active replicase of the archetypical RNA phage MS2 can be produced in a recombinant cell-free expression system. We find that the 3′ terminal fusion of antisense RNAs with a domain derived from the reverse complement of the wild type MS2 genome generates efficient templates for transcription by the MS2 replicase. The new system enables DNA-independent gene expression both in batch reactions and in microcompartments. Finally, we demonstrate that MS2-based RNA-dependent transcription-translation reactions can be used to control DNA-dependent gene expression by encoding a viral DNA-dependent RNA polymerase on a MS2 RNA template. Our study sheds light on the template requirements of the MS2 replicase and paves the way for new in vitro applications including the design of genetic circuits combining both DNA- and RNA-encoded systems.

Zusammenfassung

We present a microfluidic platform for studying structure-function relationships at the cellular level by connecting video rate live cell imaging with in situ microfluidic cryofixation and cryo-electron tomography of near natively preserved, unstained specimens. Correlative light and electron microscopy (CLEM) has been limited by the time required to transfer live cells from the light microscope to dedicated cryofixation instruments, such as a plunge freezer or high-pressure freezer. We recently demonstrated a microfluidic based approach that enables sample cryofixation directly in the light microscope with millisecond time resolution, a speed improvement of up to three orders of magnitude. Here we show that this cryofixation method can be combined with cryo-electron tomography (cryo-ET) by using Focused Ion Beam milling at cryogenic temperatures (cryo-FIB) to prepare frozen hydrated electron transparent sections. To make cryo-FIB sectioning of rapidly frozen microfluidic channels achievable, we developed a sacrificial layer technique to fabricate microfluidic devices with a PDMS bottom wall <5 µm thick. We demonstrate the complete workflow by rapidly cryo-freezing Caenorhabditis elegans roundworms L1 larvae during live imaging in the light microscope, followed by cryo-FIB milling and lift out to produce thin, electron transparent sections for cryo-ET imaging. Cryo-ET analysis of initial results show that the structural preservation of the cryofixed C. elegans was suitable for high resolution cryo-ET work. The combination of cryofixation during live imaging enabled by microfluidic cryofixation with the molecular resolution capabilities of cryo-ET offers an exciting avenue to further advance space-time correlative light and electron microscopy (st-CLEM) for investigation of biological processes at high resolution in four dimensions.

Zusammenfassung

In bottom-up synthetic biology, one of the major methodological challenges is to provide reaction spaces that mimic biological systems with regard to topology and surface functionality. Of particular interest are cell- or organelle-shaped membrane compartments, as many protein functions unfold at lipid interfaces. However, shaping artificial cell systems using materials with non-intrusive physicochemical properties, while maintaining flexible lipid interfaces relevant to the reconstituted protein systems, is not straightforward. Herein, we develop micropatterned chambers from CYTOP, a less commonly used polymer with good chemical resistance and a refractive index matching that of water. By forming a self-assembled lipid monolayer on the polymer surface, we dramatically increased the biocompatibility of CYTOP-fabricated systems. The phospholipid interface provides an excellent passivation layer to prevent protein adhesion to the hydrophobic surface, and we succeeded in cell-free protein synthesis inside the chambers. Importantly, the chambers could be sealed after loading by a lipid monolayer, providing a novel platform to study encapsulated systems. We successfully reconstituted pole-to-pole oscillations of the Escherichia coli MinDE system, which responds dramatically to compartment geometry. Furthermore, we present a simplified fabrication of our artificial cell compartments via replica molding, making it a readily accessible technique for standard cleanroom facilities.

Zusammenfassung

We engineered a synthetic temperature regulation toolbox to enable protocells to sense and respond to heat, utilizing RNA thermometers. The thermo-sensitive protocells were generated by encapsulating temperature feedback transcription/translation machinery in droplets. Based on these temperature-sensing devices, the protocells can be operated with logic AND gates, differentially processing temperature stimuli into biological signals.

Zusammenfassung

One of the most exciting discoveries in biological adhesion is the recent and counter-intuitive observation that the lifetimes of some biological adhesive bonds, called catch bonds, are enhanced by tensile mechanical force. At least two types of adhesive proteins have been shown to form catch bonds--blood proteins called selectins and a bacterial protein called FimH. Both mediate shear-enhanced adhesion, in which cells bind more strongly at high shear than at low shear. Single-molecule experiments and cell-free assays have now clearly demonstrated that catch bonds exist and mediate shear-enhanced adhesion. However, the mechanics of cellular organelles also contribute to shear-enhanced adhesion by modulating the force applied to catch bonds. This review examines how individual catch bond behavior contributes to shear-enhanced cellular adhesion for the two best-understood examples. The lessons from these systems offer design principles for understanding other types of shear-enhanced adhesion and for engineering nanostructured force-dependent adhesives out of catch bonds.

Zusammenfassung

Min protein oscillations position the bacterial cell division machinery at the cell center through a reaction‐diffusion mechanism. Here we report the successful encapsulation of the oscillating Min protein system in giant unilamellar vesicles (GUVs). Using confocal fluorescence microscopy, we identify several distinct modes of spatiotemporal patterns inside spherical GUVs. Interestingly, for osmotically deflated GUVs, we observe that the vesicle shape actively changes in concert with the Min oscillations. Our results show that the periodic relocation of Min proteins from the vesicle lumen to the membrane and back is accompanied by drastic changes in the mechanical properties of the lipid bilayer. In particular, two types of oscillating membrane‐shape changes are highlighted: (i) GUVs that repeatedly undergo fission into two connected compartments and fusion of these compartments back into a dumbbell‐shape, and (ii) GUVs that show periodic budding and subsequent merging of the buds with the mother vesicle, accompanied by an overall shape change of the vesicle, reminiscent of a bouncing ball. These findings demonstrate how reaction‐diffusion based protein self‐organization can directly yield visible mechanical effects on membrane compartments, even up to autonomous division, without the need for coupling to cytoskeletal elements. This offers fascinating perspectives for the bottom‐up design of minimal cell division.

Zusammenfassung

Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. Here, we demonstrate a general method for capturing enzyme catalysis “in action” by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis $\beta$-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s. MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data.

Zusammenfassung

© This protocol describes fabricating microfluidic devices with low X-ray background optimized for goniometer based fixed target serial crystallography. The devices are patterned from epoxy glue using soft lithography and are suitable for in situ X-ray diffraction experiments at room temperature. The sample wells are lidded on both sides with polymeric polyimide foil windows that allow diffraction data collection with low X-ray background. This fabrication method is undemanding and inexpensive. After the sourcing of a SU-8 master wafer, all fabrication can be completed outside of a cleanroom in a typical research lab environment. The chip design and fabrication protocol utilize capillary valving to microfluidically split an aqueous reaction into defined nanoliter sized droplets. This loading mechanism avoids the sample loss from channel dead-volume and can easily be performed manually without using pumps or other equipment for fluid actuation. We describe how isolated nanoliter sized drops of protein solution can be monitored in situ by dynamic light scattering to control protein crystal nucleation and growth. After suitable crystals are grown, complete X-ray diffraction datasets can be collected using goniometer based in situ fixed target serial X-ray crystallography at room temperature. The protocol provides custom scripts to process diffraction datasets using a suite of software tools to solve and refine the protein crystal structure. This approach avoids the artefacts possibly induced during cryo-preservation or manual crystal handling in conventional crystallography experiments. We present and compare three protein structures that were solved using small crystals with dimensions of approximately 10-20 µm grown in chip. By crystallizing and diffracting in situ, handling and hence mechanical disturbances of fragile crystals is minimized. The protocol details how to fabricate a custom X-ray transparent microfluidic chip suitable for in situ serial crystallography. As almost every crystal can be used for diffraction data collection, these microfluidic chips are a very efficient crystal delivery method.

Zusammenfassung

The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a $\beta$-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.

Zusammenfassung

Serial diffraction data collected at the Linac Coherent Light Source from crystalline amyloid fibrils delivered in a liquid jet show that the fibrils are well oriented in the jet. At low fibril concentrations, diffraction patterns are recorded from single fibrils; these patterns are weak and contain only a few reflections. Methods are developed for determining the orientation of patterns in reciprocal space and merging them in three dimensions. This allows the individual structure amplitudes to be calculated, thus overcoming the limitations of orientation and cylindrical averaging in conventional fibre diffraction analysis. The advantages of this technique should allow structural studies of fibrous systems in biology that are inaccessible using existing techniques.

Zusammenfassung

© 2017 The Royal Society of Chemistry. We demonstrate a novel multilayer polydimethylsiloxane (PDMS) device for selective storage and release of single emulsion droplets. Drops are captured in a microchannel cavity and can be released on-demand through a triggered surface acoustic wave pulse. The surface acoustic wave (SAW) is excited by a tapered interdigital transducer (TIDT) deposited on a piezoelectric lithium niobate (LiNbO 3 ) substrate and inverts the pressure difference across the cavity trap to push a drop out of the trap and back into the main flow channel. Droplet capture and release does not require a flow rate change, flow interruption, flow inversion or valve action and can be achieved in as fast as 20 ms. This allows both on-demand droplet capture for analysis and monitoring over arbitrary time scales, and continuous device operation with a high droplet rate of 620 drops per s. We hence decouple long-term droplet interrogation from other operations on the chip. This will ease integration with other microfluidic droplet operations and functional components.

Zusammenfassung

The construction of a minimal cell that exhibits the essential characteristics of life is a great challenge in the field of synthetic biology. Assembling a minimal cell requires multidisciplinary expertise from physics, chemistry and biology. Scientists from different backgrounds tend to define the essence of 'life' differently and have thus proposed different artificial cell models possessing one or several essential features of living cells. Using the tools and methods of molecular biology, the bottom-up engineering of a minimal cell appears in reach. However, several challenges still remain. In particular, the integration of individual sub-systems that is required to achieve a self-reproducing cell model presents a complex optimization challenge. For example, multiple self-organisation and self-assembly processes have to be carefully tuned. We review advances and developments of new methods and techniques, for cell-free protein synthesis as well as micro-fabrication, for their potential to resolve challenges and to accelerate the development of minimal cells.

Zusammenfassung

Unravelling the interaction of biological macromolecules with ligands and substrates at high spatial and temporal resolution remains a major challenge in structural biology. The development of serial crystallography methods at X-ray free-electron lasers and subsequently at synchrotron light sources allows new approaches to tackle this challenge. Here, a new polyimide tape drive designed for mix-and-diffuse serial crystallography experiments is reported. The structure of lysozyme bound by the competitive inhibitor chitotriose was determined using this device in combination with microfluidic mixers. The electron densities obtained from mixing times of 2 and 50 s show clear binding of chitotriose to the enzyme at a high level of detail. The success of this approach shows the potential for high-throughput drug screening and even structural enzymology on short timescales at bright synchrotron light sources.

Zusammenfassung

© Max O. Wiedorn et al. 2017. The success of diffraction experiments from weakly scattering samples strongly depends on achieving an optimal signal-to-noise ratio. This is particularly important in single-particle imaging experiments where diffraction signals are typically very weak and the experiments are often accompanied by significant background scattering. A simple way to tremendously reduce background scattering by placing an aperture downstream of the sample has been developed and its application in a single-particle X-ray imaging experiment at FLASH is demonstrated. Using the concept of a post-sample aperture it was possible to reduce the background scattering levels by two orders of magnitude.Diffraction experiments with weakly scattering samples often suffer from a low signal-to-noise ratio due to unwanted background scatter. Improving the signal-to-noise ratio for single-particle imaging experiments is particularly important as the diffraction signal is very weak. Here, a simple way to minimize the background scattering by placing an aperture downstream of the sample is demonstrated.

Zusammenfassung

© 2017 Elsevier B.V. We introduce a low-cost, high yield rapid fabrication method for casting COC microfluidic chips that is appropriate for academic labs and small companies. Devices are comprised of two molded pieces joined together to create a sealed device. The first piece contains the microfluidic features and the second contains the inlet and outlet manifold, a frame for rigidity and a viewing window. The microfluidic featu res are patterned using a PDMS mold that itself was replica-molded from a photoresist master. Dimensional stability of the microfluidics portion of the COC device is achieved by confining the PDMS mold in an aluminium frame. The mold for the lid is CNC milled from aluminium. Sealing the COC device is accomplished by timed immersion of the lid in a mixture of volatile and non-volatile solvents followed by application of heat and pressure. Surface treatment to render the device fluorophilic is performed using dopamine in assembled devices.

Zusammenfassung

© The Author(s) 2017. Serial femtosecond crystallography requires reliable and efficient delivery of fresh crystals across the beam of an X-ray free-electron laser over the course of an experiment. We introduce a double-flow focusing nozzle to meet this challenge, with significantly reduced sample consumption, while improving jet stability over previous generations of nozzles. We demonstrate its use to determine the first room-temperature structure of RNA polymerase II at high resolution, revealing new structural details. Moreover, the double flow-focusing nozzles were successfully tested with three other protein samples and the first room temperature structure of an extradiol ring-cleaving dioxygenase was solved by utilizing the improved operation and characteristics of these devices.

Zusammenfassung

© 2016 Robin Schubert et al. Many biochemical processes take place on timescales ranging from femto-seconds to seconds. Accordingly, any time-resolved experiment must be matched to the speed of the structural changes of interest. Therefore, the timescale of interest defines the requirements of the X-ray source, instrumentation and data-collection strategy. In this study, a minimalistic approach for in situ crystallization is presented that requires only a few microlitres of sample solution containing a few hundred crystals. It is demonstrated that complete diffraction data sets, merged from multiple crystals, can be recorded within only a few minutes of beamtime and allow high-resolution structural information of high quality to be obtained with a temporal resolution of 40 ms. Global and site-specific radiation damage can be avoided by limiting the maximal dose per crystal to 400 kGy. Moreover, analysis of the data collected at higher doses allows the time-resolved observation of site-specific radiation damage. Therefore, our approach is well suited to observe structural changes and possibly enzymatic reactions in the low-millisecond regime.

Zusammenfassung

©2016 Optical Society of America. Reliable sample delivery is essential to biological imaging using X-ray Free Electron Lasers (XFELs). Continuous injection using the Gas Dynamic Virtual Nozzle (GDVN) has proven valuable, particularly for time-resolved studies. However, many important aspects of GDVN functionality have yet to be thoroughly understood and/or refined due to fabrication limitations. We report the application of 2-photon polymerization as a form of high-resolution 3D printing to fabricate high-fidelity GDVNs with submicron resolution. This technique allows rapid prototyping of a wide range of different types of nozzles from standard CAD drawings and optimization of crucial dimensions for optimal performance. Three nozzles were tested with pure water to determine general nozzle performance and reproducibility, with nearly reproducible off-axis jetting being the result. X-ray tomography and index matching were successfully used to evaluate the interior nozzle structures and identify the cause of off-axis jetting. Subsequent refinements to fabrication resulted in straight jetting. A performance test of printed nozzles at an XFEL provided high quality femtosecond diffraction patterns.

Zusammenfassung

© 2016, EDP Sciences and Springer. We investigate the Belousov–Zhabotinsky (BZ) reaction in an attempt to establish a basis for computation using chemical oscillators coupled via inhibition. The system consists of BZ droplets suspended in oil. Interdrop coupling is governed by the non-polar communicator of inhibition, Br 2 . We consider a linear arrangement of three droplets to be a NOR gate, where the center droplet is the output and the other two are inputs. Oxidation spikes in the inputs, which we define to be TRUE, cause a delay in the next spike of the output, which we read to be FALSE. Conversely, when the inputs do not spike (FALSE) there is no delay in the output (TRUE), thus producing the behavior of a NOR gate. We are able to reliably produce NOR gates with this behavior in microfluidic experiment.

Zusammenfassung

The motility of cilia and flagella is driven by thousands of dynein motors that hydrolyze adenosine triphosphate (ATP). Despite decades of genetic, biochemical, structural and biophysical studies, some aspects of ciliary motility remain elusive, such as the regulation of beating patterns and the energetic efficiency of these nanomachines. Here, we introduce an experimental method to measure ATP consumption of actively beating axonemes on a single-cell level. We encapsulated individual sea urchin sperm with demembranated flagellum inside water-in-oil emulsion droplets and measured the axonemes ATP consumption by monitoring fluorescence intensity of a fluorophore-coupled reporter system for ATP turnover in the droplet. Concomitant phase contrast imaging allowed us to extract a linear dependence between the ATP consumption rate and the flagellar beating frequency, with \~2.3e5 ATP molecules consumed per beat of a demembranated flagellum. Increasing the viscosity of the aqueous medium led to modified beating waveforms of the axonemes and to higher energy consumption per beat cycle. Our single-cell experimental platform provides both new insights into the beating mechanism of flagella and a powerful tool for future studies.

Zusammenfassung

Serial femtosecond crystallography (SFX) using X-ray Free-Electron Lasers (XFELs) allows for room temperature protein structure determination without evidence of conventional radiation damage. In this method, a liquid suspension of protein microcrystals can be delivered to the X-ray beam in vacuum as a micro-jet, which replenishes the crystals at a rate that exceeds the current XFEL pulse repetition rate. Gas dynamic virtual nozzles produce the required micrometer-sized streams by the focusing action of a coaxial sheath gas and have been shown to be effective for SFX experiments. Here, we describe the design and characterization of such nozzles assembled from ceramic micro-injection molded outer gas-focusing capillaries. Trends of the emitted jet diameter and jet length as a function of supplied liquid and gas flow rates are measured by a fast imaging system. The observed trends are explained by derived relationships considering choked gas flow and liquid flow conservation. Finally, the performance of these nozzles in a SFX experiment is presented, including an analysis of the observed background.

Zusammenfassung

\textlessp\textgreaterLipidic cubic phases (LCPs) have emerged as successful matrixes for the crystallization of membrane proteins. Moreover, the viscous LCP also provides a highly effective delivery medium for serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs). Here, the adaptation of this technology to perform serial millisecond crystallography (SMX) at more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates the need for difficult handling of individual crystals and allows for data collection at room temperature. The technology is demonstrated by solving a structure of the light-driven proton-pump bacteriorhodopsin (bR) at a resolution of 2.4 \AA. The room-temperature structure of bR is very similar to previous cryogenic structures but shows small yet distinct differences in the retinal ligand and proton-transfer pathway.\textless/p\textgreater

Zusammenfassung

X-ray free-electron lasers (XFELs) offer a new avenue to the structural probing of complex materials, including biomolecules. Delivery of precious sample to the XFEL beam is a key consideration, as the sample of interest must be serially replaced after each destructive pulse. The fixed-target approach to sample delivery involves depositing samples on a thin-film support and subsequent serial introduction via a translating stage. Some classes of biological materials, including two-dimensional protein crystals, must be introduced on fixed-target supports, as they require a flat surface to prevent sample wrinkling. A series of wafer and transmission electron microscopy (TEM)-style grid supports constructed of low- Z plastic have been custom-designed and produced. Aluminium TEM grid holders were engineered, capable of delivering up to 20 different conventional or plastic TEM grids using fixed-target stages available at the Linac Coherent Light Source (LCLS). As proof-of-principle, X-ray diffraction has been demonstrated from two-dimensional crystals of bacteriorhodopsin and three-dimensional crystals of anthrax toxin protective antigen mounted on these supports at the LCLS. The benefits and limitations of these low- Z fixed-target supports are discussed; it is the authors' belief that they represent a viable and efficient alternative to previously reported fixed-target supports for conducting diffraction studies with XFELs.

Zusammenfassung

Serial femtosecond crystallography using ultrashort pulses from x-ray free electron lasers (XFELs) enables studies of the light-triggered dynamics of biomolecules. We used microcrystals of photoactive yellow protein (a bacterial blue light photoreceptor) as a model system and obtained high-resolution, time-resolved difference electron density maps of excellent quality with strong features; these allowed the determination of structures of reaction intermediates to a resolution of 1.6 angstroms. Our results open the way to the study of reversible and nonreversible biological reactions on time scales as short as femtoseconds under conditions that maximize the extent of reaction initiation throughout the crystal.

Zusammenfassung

Alan Turing, in "The Chemical Basis of Morphogenesis" Turing AM (1952) Philos Trans R Soc Lond 237(641):37-72, described how, in circular arrays of identical biological cells, diffusion can interact with chemical reactions to generate up to six periodic spatiotemporal chemical structures. Turing proposed that one of these structures, a stationary pattern with a chemically determined wavelength, is responsible for differentiation. We quantitatively test Turing's ideas in a cellular chemical system consisting of an emulsion of aqueous droplets containing the Belousov-Zhabotinsky oscillatory chemical reactants, dispersed in oil, and demonstrate that reaction-diffusion processes lead to chemical differentiation, which drives physical morphogenesis in chemical cells. We observe five of the six structures predicted by Turing. In 2D hexagonal arrays, a seventh structure emerges, incompatible with Turing's original model, which we explain by modifying the theory to include heterogeneity.

Zusammenfassung

We measured the nucleation rates of lysozyme protein crystals using microfluidically produced emulsion drops containing supersaturated protein solution. The technique involves quenching several thousand independent nanoliter drops by rapidly lowering the temperature and then counting the number of drops that have not nucleated as a function of time at constant temperature. We fit the number distribution to a theoretical model developed by Pound and La Mer (J. Am. Chem. Soc. 1952, 74, 2323−2332) for heterogeneous nucleation and extract two nucleation rates and the number of nucleation sites per drop. We describe the technique in detail and present our analysis of the measured nucleation rates within the context of Classical Nucleation Theory, which adequately describes our observations. Of the two nucleation rates, one is a slow rate that varies with temperature and one is a fast rate independent of temperature. The nucleation barrier and kinetic prefactors are obtained for each rate. Notably, there is no detectable barrier for the fast rate. Both rates are inconsistent with the process of homogeneous nucleation and are consistent with heterogeneous nucleation.

Zusammenfassung

An emulsion-based serial crystallographic technology has been developed, in which nanolitre-sized droplets of protein solution are encapsulated in oil and stabilized by surfactant. Once the first crystal in a drop is nucleated, the small volume generates a negative feedback mechanism that lowers the supersaturation. This mechanism is exploited to produce one crystal per drop. Diffraction data are measured, one crystal at a time, from a series of room-temperature crystals stored on an X-ray semi-transparent microfluidic chip, and a 93\% complete data set is obtained by merging single diffraction frames taken from different unoriented crystals. As proof of concept, the structure of glucose isomerase was solved to 2.1 \AA, demonstrating the feasibility of high-throughput serial X-ray crystallography using synchrotron radiation.

Zusammenfassung

We visualize birefringence in microliter sample volumes using a microfluidic dialysis chip optimized for cross polarization microscopy. The chip is composed of two overlapping polydimethylsiloxane (PDMS) channels separated by a commercial cellulose ester membrane. Buffer exchange in the sample chamber is achieved within minutes by dialyzing under continuous reservoir flow. Using fd virus as a birefringent model system, we monitor the fd virus isotropic to liquid crystal phase transition as a function of ionic strength. We show that the reorientation of the fd virus spans a few tens of seconds, indicative of fast ion exchange across the membrane. Complete phase separation reorganization takes minutes to hours as it involves diffusive virus mass transport within the storage chamber.

Zusammenfassung

With remarkable precision and reproducibility, cells orchestrate the cooperative action of thousands of nanometre-sized molecular motors to carry out mechanical tasks at much larger length scales, such as cell motility, division and replication. Besides their biological importance, such inherently non-equilibrium processes suggest approaches for developing biomimetic active materials from microscopic components that consume energy to generate continuous motion. Being actively driven, these materials are not constrained by the laws of equilibrium statistical mechanics and can thus exhibit sought-after properties such as autonomous motility, internally generated flows and self-organized beating. Here, starting from extensile microtubule bundles, we hierarchically assemble far-from-equilibrium analogues of conventional polymer gels, liquid crystals and emulsions. At high enough concentration, the microtubules form a percolating active network characterized by internally driven chaotic flows, hydrodynamic instabilities, enhanced transport and fluid mixing. When confined to emulsion droplets, three-dimensional networks spontaneously adsorb onto the droplet surfaces to produce highly active two-dimensional nematic liquid crystals whose streaming flows are controlled by internally generated fractures and self-healing, as well as unbinding and annihilation of oppositely charged disclination defects. The resulting active emulsions exhibit unexpected properties, such as autonomous motility, which are not observed in their passive analogues. Taken together, these observations exemplify how assemblages of animate microscopic objects exhibit collective biomimetic properties that are very different from those found in materials assembled from inanimate building blocks, challenging us to develop a theoretical framework that would allow for a systematic engineering of their far-from-equilibrium material properties.

Zusammenfassung

Microfluidic devices can be molded easily from PDMS using soft lithography. However, the softness of the resulting microchannels makes it difficult to photolithographically pattern their surface properties, as is needed for applications such as double emulsification. We introduce a new patterning method for PDMS devices, using integrated oxygen reservoirs fabricated simultaneously with the microfluidic channels, which serve as "chemo-masks". Oxygen diffuses through the PDMS to the nearby channel segments and there inhibits functional polymer growth; by placement of the chemo-masks, we thus control the polymerization pattern. This patterning method is simple, scalable, and compatible with a variety of surface chemistries.