Systems and Methods for Continuous Evolution of Enzymes.

Directed evolution generates novel biomolecules with desired functions by iteratively diversifying the genetic sequence of wildtype biomolecules, relaying the genetic information to the molecule with function, and selecting the variants that progresses towards the properties of interest. While traditional directed evolution consumes significant labor and time for each step, continuous evolution seeks to automate all steps so directed evolution can proceed with minimum human intervention and dramatically shortened time. A major application of continuous evolution is the generation of novel enzymes, which catalyze reactions under conditions that are not favorable to their wildtype counterparts, or on altered substrates. The challenge to continuously evolve enzymes lies in automating sufficient, unbiased gene diversification, providing selection for a wide array of reaction types, and linking the genetic information to the phenotypic function. Over years of development, continuous evolution has accumulated versatile strategies to address these challenges, enabling its use as a general tool for enzyme engineering. As the capability of continuous evolution continues to expand, its impact will increase across various industries. In this review, we summarize the working mechanisms of recently developed continuous evolution strategies, discuss examples of their applications focusing on enzyme evolution, and point out their limitations and future directions.

Work hardening in colloidal crystals.

Colloidal crystals exhibit interesting properties1-4 that are in many ways analogous to their atomic counterparts. They have the same crystal structures2,5-7, undergo the same phase transitions8-10, and possess the same crystallographic defects11-14. In contrast to these structural properties, the mechanical properties of colloidal crystals are quite different from those of atomic systems. For example, unlike in atomic systems, the elasticity of hard-sphere colloidal crystals is purely entropic15; as a result, they are so soft that they can be melted just by stirring16,17. Moreover, crystalline materials deform plastically when subjected to increasing shear and become stronger because of the ubiquitous process of work hardening18; but this has so far never been observed in colloidal crystals, to our knowledge. Here we show that hard-sphere colloidal crystals exhibit work hardening. Moreover, despite their softness, the shear strength of colloidal crystals can increase and approach the theoretical limit for crystals, a value reached in very few other materials so far. We use confocal microscopy to show that the strength of colloidal crystals increases with dislocation density, and ultimately reaches the classic Taylor scaling behaviour for atomic materials19-21, although hard-sphere interactions lack the complexity of atomic interactions. We demonstrate that Taylor hardening arises through the formation of dislocation junctions22. The Taylor hardening regime, however, is established only after a transient phase, and it ceases when the colloidal crystals become so hard that the strain is localized within a thin boundary layer in which slip results from an unconventional motion of dislocations. The striking resemblance between colloidal and atomic crystals, despite the many orders of magnitude difference in particle size and shear modulus, demonstrates the universality of work hardening.

High-throughput direct screening of restriction endonuclease using a microfluidic fluorescence-activated drop sorter based on the SOS response in Escherichia coli.

A restriction endonuclease (RE) is an enzyme that can recognize a specific DNA sequence and cleave that DNA into fragments with double-stranded breaks. This sequence-specific cleaving ability and its ease of use have made REs commonly used tools in molecular biology since their first isolation and characterization in 1970s. While artificial REs still face many challenges in large-scale synthesis and precise activity control for practical use, searching for new REs in natural samples remains a viable route to expanding the RE pool for fundamental research and industrial applications. In this paper, we propose a new strategy to search for REs in an efficient manner. We constructed a host bacterial cell to link the genotype of REs to the phenotype of ß-galactosidase expression based on the bacterial SOS response, and used a high-throughput microfluidic platform to isolate, detect and sort the REs in microfluidic drops at a frequency of ∼800 drops per second. We employed this strategy to screen for the XbaI gene from the constructed libraries of varied sizes. In a single round of sorting, a 90-fold target enrichment was achieved within 1 h. Compared to conventional RE-screening methods, the direct screening approach that we propose excels at efficient search of desirable REs in natural samples - especially unculturable samples - and can be tailored to high-throughput screening of a wide range of genotoxic targets.

Polysaccharide functionalization reduces lipid vesicle stiffness.

The biophysical properties of lipid vesicles are important for their stability and integrity, key parameters that control the performance when these vesicles are used for drug delivery. The vesicle properties are determined by the composition of lipids used to form the vesicle. However, for a given lipid composition, they can also be tailored by tethering polymers to the membrane. Typically, synthetic polymers like polyethyleneglycol are used to increase vesicle stability, but the use of polysaccharides in this context is much less explored. Here, we report a general method for functionalizing lipid vesicles with polysaccharides by binding them to cholesterol. We incorporate the polysaccharides on the outer membrane leaflet of giant unilamellar vesicles (GUVs) and investigate their effect on membrane mechanics using micropipette aspiration. We find that the presence of the glycolipid functionalization produces an unexpected softening of GUVs with fluid-like membranes. By contrast, the functionalization of GUVs with polyethylene glycol does not reduce their stretching modulus. This work provides the potential means to study membrane-bound meshworks of polysaccharides similar to the cellular glycocalyx; moreover, it can be used for tuning the mechanical properties of drug delivery vehicles.

Ultrasoft silicone gels with tunable refractive index for traction force microscopy.

We formulate and characterize silicone gels near the gelation threshold with tunable refractive index, 1.4 < n < 1.49, and small viscoelastic moduli, G'∼1 Pa, for use in traction force microscopy. The near-critical gels have low-frequency storage plateau moduli between 50 Pa and 1 Pa, with loss moduli that are more than fifty times lower at low frequencies. The gels are linearly elastic up to strains of at least 50%. The refractive index of the gel is tuned to eliminate spherical aberrations during confocal imaging thereby minimizing signal loss when imaging through thick gel substrates. We also develop an index-matched colloidal particle, stabilized by a silicone brush, that can be dispersed throughout the gel. These particles can be used to determine the deformation of the gel. The combination of mechanical and optical properties of these near-critical gels extends the lower limit of stresses that can be measured with traction force microscopy to single mPa values, while minimizing optical aberrations.

Thin adhesive oil films lead to anomalously stable mixtures of water in oil.

Oil and water can only be mixed by dispersing droplets of one fluid in the other. When two droplets approach one another, the thin film that separates them invariably becomes unstable, causing the droplets to coalesce. The only known way to avoid this instability is through addition of a third component, typically a surfactant, which stabilizes the thin film at its equilibrium thickness. We report the observation that a thin fluid film of oil separating two water droplets can lead to an adhesive interaction between the droplets. Moreover, this interaction prevents their coalescence over timescales of several weeks, without the use of any surfactant or solvent.

Surface-Functionalized Microgels as Artificial Antigen-Presenting Cells to Regulate Expansion of T Cells.

Artificial antigen-presenting cells (aAPCs) are currently used to manufacture T cells for adoptive therapy in cancer treatment, but a readily tunable and modular system can enable both rapid T cell expansion and control over T cell phenotype. Here, it is shown that microgels with tailored surface biochemical properties can serve as aAPCs to mediate T cell activation and expansion. Surface functionalization of microgels is achieved via layer-by-layer coating using oppositely charged polymers, forming a thin but dense polymer layer on the surface. This facile and versatile approach is compatible with a variety of coating polymers and allows efficient and flexible surface-specific conjugation of defined peptides or proteins. The authors demonstrate that tethering appropriate stimulatory ligands on the microgel surface efficiently activates T cells for polyclonal and antigen-specific expansion. The expansion, phenotype, and functional outcome of primary mouse and human T cells can be regulated by modulating the concentration, ratio, and distribution of stimulatory ligands presented on microgel surfaces as well as the stiffness and viscoelasticity of the microgels.

Toward the quantification of α-synuclein aggregates with digital seed amplification assays.

The quantification and characterization of aggregated α-synuclein in clinical samples offer immense potential toward diagnosing, treating, and better understanding neurodegenerative synucleinopathies. Here, we developed digital seed amplification assays to detect single α-synuclein aggregates by partitioning the reaction into microcompartments. Using pre-formed α-synuclein fibrils as reaction seeds, we measured aggregate concentrations as low as 4 pg/mL. To improve our sensitivity, we captured aggregates on antibody-coated magnetic beads before running the amplification reaction. By first characterizing the pre-formed fibrils with transmission electron microscopy and size exclusion chromatography, we determined the specific aggregates targeted by each assay platform. Using brain tissue and cerebrospinal fluid samples collected from patients with Parkinson's Disease and multiple system atrophy, we demonstrated that the assay can detect endogenous pathological α-synuclein aggregates. Furthermore, as another application for these assays, we studied the inhibition of α-synuclein aggregation in the presence of small-molecule inhibitors and used a custom image analysis pipeline to quantify changes in aggregate growth and filament morphology.

Development and future of droplet microfluidics.

Over the past two decades, advances in droplet-based microfluidics have facilitated new approaches to process and analyze samples with unprecedented levels of precision and throughput. A wide variety of applications has been inspired across multiple disciplines ranging from materials science to biology. Understanding the dynamics of droplets enables optimization of microfluidic operations and design of new techniques tailored to emerging demands. In this review, we discuss the underlying physics behind high-throughput generation and manipulation of droplets. We also summarize the applications in droplet-derived materials and droplet-based lab-on-a-chip biotechnology. In addition, we offer perspectives on future directions to realize wider use of droplet microfluidics in industrial production and biomedical analyses.

Multichannel Multijunction Droplet Microfluidic Device to Synthesize Hydrogel Microcapsules with Different Core-Shell Structures and Adjustable Core Positions.

Core-shell hydrogel microcapsules have sparked great interest due to their unique characteristics and prospective applications in the medical, pharmaceutical, and cosmetic fields. However, complex synthetic procedures and expensive costs have limited their practical application. Herein, we designed and prepared several multichannel and multijunctional droplet microfluidic devices based on soft lithography for the effective synthesis of core-shell hydrogel microcapsules for different purposes. Additionally, two different cross-linking processes (ultraviolet (UV) exposure and interfacial polymerization) were used to synthesize different types of core-shell structured hydrogel microcapsules. Hydrogel microcapsules with gelatin methacryloyl (GelMA) as the core and polyacrylamide (PAM) as the thin shell were synthesized using UV cross-linking. Using an interfacial polymerization process, another core-shell structured microcapsule with GelMA as the core and Ca2+ cross-linked alginate with polyethylenimine (PEI) as the shell was constructed, and the core diameter and total droplet diameter were flexibly controlled by carving. Noteworthy, these hydrogel microcapsules exhibit stimuli-responsiveness and controlled release ability. Overall, a novel technique was developed to successfully synthesize various hydrogel microcapsules with core-shell microstructures. The hydrogel microcapsules possess a multilayered structure that facilitates the coassembly of cells and drugs, as well as the layered assembly of multiple drugs, to develop synergistic therapeutic regimens. These adaptable and controllable hydrogel microdroplets shall held great promise for multicell or multidrug administration as well as for high-throughput drug screening.

High-sensitivity whole-genome recovery of single viral species in environmental samples.

Characterizing unknown viruses is essential for understanding viral ecology and preparing against viral outbreaks. Recovering complete genome sequences from environmental samples remains computationally challenging using metagenomics, especially for low-abundance species with uneven coverage. This work presents a method for reliably recovering complete viral genomes from complex environmental samples. Individual genomes are encapsulated into droplets and amplified using multiple displacement amplification. A novel gene detection assay, which employs an RNA-based probe and an exonuclease, selectively identifies droplets containing the target viral genome. Labeled droplets are sorted using a microfluidic sorter, and genomes are extracted for sequencing. Validation experiments using a sewage sample spiked with two known viruses demonstrate the method's efficacy. We achieve 100% recovery of the spiked-in SV40 (Simian virus 40, 5243bp) genome sequence with uniform coverage distribution, and approximately 99.4% for the larger HAd5 genome (Human Adenovirus 5, 35938bp). Notably, genome recovery is achieved with as few as one sorted droplet, which enables the recovery of any desired genomes in complex environmental samples, regardless of their abundance. This method enables targeted characterizations of rare viral species and whole-genome amplification of single genomes for accessing the mutational profile in single virus genomes, contributing to an improved understanding of viral ecology.

Dislocation interactions during plastic relaxation of epitaxial colloidal crystals.

The severe difficulty to resolve simultaneously both the macroscopic deformation process and the dislocation dynamics on the atomic scale limits our understanding of crystal plasticity. Here we use colloidal crystals, imaged on the single particle level by high-speed three-dimensional (3D) confocal microscopy, and resolve in real-time both the relaxation of the epitaxial misfit strain and the accompanying evolution of dislocations. We show how dislocation interactions give rise to the formation of complex dislocation networks in 3D and to unexpectedly sharp plastic relaxation. The sharp relaxation is facilitated by attractive interactions that promote the formation of new dislocations that are more efficient in mediating strain. Dislocation networks form fragmented structures, as dislocation growth is blocked by either attractive interactions, which result in the formation of sessile dislocation junctions, or by repulsion from perpendicular segments. The strength of these blocking mechanisms decreases with the thickness of the crystal film. These results reveal the critical role of dislocation interactions in plastic deformation of thin films and can be readily generalized from the colloidal to the atomic scale.

Anomalous crystalline ordering of particles in a viscoelastic fluid under high shear.

Addition of particles to a viscoelastic suspension dramatically alters the properties of the mixture, particularly when it is sheared or otherwise processed. Shear-induced stretching of the polymers results in elastic stress that causes a substantial increase in measured viscosity with increasing shear, and an attractive interaction between particles, leading to their chaining. At even higher shear rates, the flow becomes unstable, even in the absence of particles. This instability makes it very difficult to determine the properties of a particle suspension. Here, we use a fully immersed parallel plate geometry to measure the high-shear-rate behavior of a suspension of particles in a viscoelastic fluid. We find an unexpected separation of the particles within the suspension resulting in the formation of a layer of particles in the center of the cell. Remarkably, monodisperse particles form a crystalline layer which dramatically alters the shear instability. By combining measurements of the velocity field and torque fluctuations, we show that this solid layer disrupts the flow instability and introduces a single-frequency component to the torque fluctuations that reflects a dominant velocity pattern in the flow. These results highlight the interplay between particles and a suspending viscoelastic fluid at very high shear rates.

The liquid-to-solid transition of FUS is promoted by the condensate surface.

A wide range of macromolecules can undergo phase separation, forming biomolecular condensates in living cells. These membraneless organelles are typically highly dynamic, formed reversibly, and carry out essential functions in biological systems. Crucially, however, a further liquid-to-solid transition of the condensates can lead to irreversible pathological aggregation and cellular dysfunction associated with the onset and development of neurodegenerative diseases. Despite the importance of this liquid-to-solid transition of proteins, the mechanism by which it is initiated in normally functional condensates is unknown. Here we show, by measuring the changes in structure, dynamics, and mechanics in time and space, that single-component FUS condensates do not uniformly convert to a solid gel, but rather that liquid and gel phases coexist simultaneously within the same condensate, resulting in highly inhomogeneous structures. Furthermore, our results show that this transition originates at the interface between the condensate and the dilute continuous phase, and once initiated, the gelation process propagates toward the center of the condensate. To probe such spatially inhomogeneous rheology during condensate aging, we use a combination of established micropipette aspiration experiments together with two optical techniques, spatial dynamic mapping and reflective confocal dynamic speckle microscopy. These results reveal the importance of the spatiotemporal dimension of the liquid-to-solid transition and highlight the interface of biomolecular condensates as a critical element in driving pathological protein aggregation.

Droplet-based high-throughput single microbe RNA sequencing by smRandom-seq.

Bacteria colonize almost all parts of the human body and can differ significantly. However, the population level transcriptomics measurements can only describe the average bacteria population behaviors, ignoring the heterogeneity among bacteria. Here, we report a droplet-based high-throughput single-microbe RNA-seq assay (smRandom-seq), using random primers for in situ cDNA generation, droplets for single-microbe barcoding, and CRISPR-based rRNA depletion for mRNA enrichment. smRandom-seq showed a high species specificity (99%), a minor doublet rate (1.6%), a reduced rRNA percentage (32%), and a sensitive gene detection (a median of ~1000 genes per single E. coli). Furthermore, smRandom-seq successfully captured transcriptome changes of thousands of individual E. coli and discovered a few antibiotic resistant subpopulations displaying distinct gene expression patterns of SOS response and metabolic pathways in E. coli population upon antibiotic stress. smRandom-seq provides a high-throughput single-microbe transcriptome profiling tool that will facilitate future discoveries in microbial resistance, persistence, microbe-host interaction, and microbiome research.

Positional influence on cellular transcriptional identity revealed through spatially segmented single-cell transcriptomics.

Single-cell RNA sequencing (scRNA-seq) is a powerful technique for describing cell states. Identifying the spatial arrangement of these states in tissues remains challenging, with the existing methods requiring niche methodologies and expertise. Here, we describe segmentation by exogenous perfusion (SEEP), a rapid and integrated method to link surface proximity and environment accessibility to transcriptional identity within three-dimensional (3D) disease models. The method utilizes the steady-state diffusion kinetics of a fluorescent dye to establish a gradient along the radial axis of disease models. Classification of sample layers based on dye accessibility enables dissociated and sorted cells to be characterized by transcriptomic and regional identities. Using SEEP, we analyze spheroid, organoid, and in vivo tumor models of high-grade serous ovarian cancer (HGSOC). The results validate long-standing beliefs about the relationship between cell state and position while revealing new concepts regarding how spatially unique microenvironments influence the identity of individual cells within tumors.

Rock-on-a-chip: "Seeing" the association/disassociation of an adaptive polymer in solutions flowing through porous media.

The flow and transport of polymer solutions through porous media are ubiquitous in myriad scientific and engineering applications. With escalating interest in adaptive polymers, understanding the flow dynamics of their solutions is indispensable (yet lacking). Here, the hydrophobic-effect-driven reversible associations in a self-adaptive polymer (SAP) solution and its flow characteristics in a microfluidic-based "rock-on-a-chip" device have been analyzed. The hydrophobic aggregates were fluorescent labeled; this enabled a direct visualization of the in situ association/disassociation of the polymer supramolecular assemblies in pore spaces and throats. Furthermore, the influence of this adaptation on the macroscopic flow behavior of the SAP solution was analyzed by comparing its flow with that of two partially-hydrolyzed polyacrylamide (the molecular weight (MW)-equivalent HPAM-1 and ultrahigh-MW HPAM-2) solutions in the semi-dilute regime with similar initial viscosities. At low flow rates (with shear predominance), the SAP solution showed a low shear viscosity compared to HPAM-1, indicating a higher shear susceptibility for association than chain entanglement. Although the SAP exhibited the same elastic instability as the non-adaptive polymers above a threshold flow rate, the adaptable structure of the former advanced the onset of its viscoelastic-governed flow, providing a stronger flow resistance, possibly through an extension resistance. Furthermore, 3D-media analysis indicated that the reversible association/disassociation of SAP increased the accessible pore space during nonaqueous-liquid displacement, facilitating oil production.

Melting of a macroscale binary Coulombic crystal.

The question of melting has been addressed theoretically and experimentally for two-dimensional crystals in thermal equilibrium. However, as it pertains to out-of-equilibrium systems, the question is unresolved. Here, we present a platform to study the melting of a two-dimensional, binary Coulombic crystal composed of equal numbers of nylon and polytetrafluoroethylene (PTFE) beads that measure a couple of millimeters in diameter. The beads are tribocharged-nylon positively and PTFE negatively-and they experience long-range electrostatic interactions. They form a square crystal in which nylon and PTFE beads sit at alternating sites on a checkerboard lattice. We melt the crystal by agitating the dish in which it resides using an orbital shaker. We compare the melting behavior of the crystal without impurities to that of the crystal with impurities, where we use gold-coated nylon beads as impurities because they tribocharge negligibly. Our results reveal that impurities do not influence the melting of the crystal. Instead, the crystal undergoes shear-induced melting, beginning from its edges, due to its collisions with the dish. As a result of repetitive collisions, the beads acquire kinetic energy, undergo rearrangements, and become disordered. Unlike most examples of shear-induced melting, portions of the crystal remain locally ordered given the persistence of electrostatic interactions and the occurrence of some collisions that are favorable to ordering clusters of beads. Our work clarifies the melting behavior of sheared crystals whose constituents have persistent long-range interactions. It may prove valuable in determining the conditions under which such materials are immune to disorder.

Spatially non-uniform condensates emerge from dynamically arrested phase separation.

The formation of biomolecular condensates through phase separation from proteins and nucleic acids is emerging as a spatial organisational principle used broadly by living cells. Many such biomolecular condensates are not, however, homogeneous fluids, but possess an internal structure consisting of distinct sub-compartments with different compositions. Notably, condensates can contain compartments that are depleted in the biopolymers that make up the condensate. Here, we show that such double-emulsion condensates emerge via dynamically arrested phase transitions. The combination of a change in composition coupled with a slow response to this change can lead to the nucleation of biopolymer-poor droplets within the polymer-rich condensate phase. Our findings demonstrate that condensates with a complex internal architecture can arise from kinetic, rather than purely thermodynamic driving forces, and provide more generally an avenue to understand and control the internal structure of condensates in vitro and in vivo.

Stimuli-Triggered Multishape, Multimode, and Multistep Deformations Designed by Microfluidic 3D Droplet Printing.

Elastomers generally possess low Young's modulus and high failure strain, which are widely used in soft robots and intelligent actuators. However, elastomers generally lack diverse functionalities, such as stimulated shape morphing, and a general strategy to implement these functionalities into elastomers is still challenging. Here, a microfluidic 3D droplet printing platform is developed to design composite elastomers architected with arrays of functional droplets. Functional droplets with controlled size, composition, position, and pattern are designed and implemented in the composite elastomers, imparting functional performances to the systems. The composited elastomers are sensitive to stimuli, such as solvent, temperature, and light, and are able to demonstrate multishape (bow- and S-shaped), multimode (gradual and sudden), and multistep (one- and two-step) deformations. Based on the unique properties of droplet-embedded composite elastomers, a variety of stimuli-responsive systems are developed, including designable numbers, biomimetic flowers, and soft robots, and a series of functional performances are achieved, presenting a facile platform to impart diverse functionalities into composite elastomers by microfluidic 3D droplet printing.