Thermostable in vitro transcription-translation compatible with microfluidic droplets.

BACKGROUND: In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures. OBJECTIVES: Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets. RESULTS: We produced cell-free extracts from Thermus thermophilus for in vitro translation including thermostable enzymatic cascades for energy regeneration and a moderately thermostable RNA polymerase for transcription, which ultimately limited the temperature of protein synthesis. The yield was comparable or superior to other thermostable in vitro expression systems, while the preparation procedure is much simpler and can be suited to different Thermus thermophilus strains. Furthermore, these extracts have enabled in vitro expression in microfluidic droplets at high temperatures for the first time. CONCLUSIONS: Cell-free extracts from Thermus thermophilus represent a simpler alternative to heavily optimized or pure component thermostable in vitro expression systems. Moreover, due to their compatibility with droplet microfluidics and enzyme assays at high temperatures, the reported system represents a convenient gateway for enzyme screening at higher temperatures with ultrahigh-throughput.

Deep learning-assisted concentration gradient generation for the study of 3D cell cultures in hydrogel beads of varying stiffness.

The study of dose-response relationships underpins analytical biosciences. Droplet microfluidics platforms can automate the generation of microreactors encapsulating varying concentrations of an assay component, providing datasets across a large chemical space in a single experiment. A classical method consists in varying the flow rate of multiple solutions co-flowing into a single microchannel (producing different volume fractions) before encapsulating the contents into water-in-oil droplets. This process can be automated through controlling the pumping elements but lacks the ability to adapt to unpredictable experimental scenarios, often requiring constant human supervision. In this paper, we introduce an image-based, closed-loop control system for assessing and adjusting volume fractions, thereby generating unsupervised, uniform concentration gradients. We trained a shallow convolutional neural network to assess the position of the laminar flow interface between two co-flowing fluids and used this model to adjust flow rates in real-time. We apply the method to generate alginate microbeads in which HEK293FT cells could grow in three dimensions. The stiffnesses ranged from 50 Pa to close to 1 kPa in Young modulus and were encoded with a fluorescent marker. We trained deep learning models based on the YOLOv4 object detector to efficiently detect both microbeads and multicellular spheroids from high-content screening images. This allowed us to map relationships between hydrogel stiffness and multicellular spheroid growth.

Droplet-based methodology for investigating bacterial population dynamics in response to phage exposure.

An alarming rise in antimicrobial resistance worldwide has spurred efforts into the search for alternatives to antibiotic treatments. The use of bacteriophages, bacterial viruses harmless to humans, represents a promising approach with potential to treat bacterial infections (phage therapy). Recent advances in microscopy-based single-cell techniques have allowed researchers to develop new quantitative methodologies for assessing the interactions between bacteria and phages, especially the ability of phages to eradicate bacterial pathogen populations and to modulate growth of both commensal and pathogen populations. Here we combine droplet microfluidics with fluorescence time-lapse microscopy to characterize the growth and lysis dynamics of the bacterium Escherichia coli confined in droplets when challenged with phage. We investigated phages that promote lysis of infected E. coli cells, specifically, a phage species with DNA genome, T7 (Escherichia virus T7) and two phage species with RNA genomes, MS2 (Emesvirus zinderi) and Qß (Qubevirus durum). Our microfluidic trapping device generated and immobilized picoliter-sized droplets, enabling stable imaging of bacterial growth and lysis in a temperature-controlled setup. Temporal information on bacterial population size was recorded for up to 25 h, allowing us to determine growth rates of bacterial populations and helping us uncover the extent and speed of phage infection. In the long-term, the development of novel microfluidic single-cell and population-level approaches will expedite research towards fundamental understanding of the genetic and molecular basis of rapid phage-induced lysis and eco-evolutionary aspects of bacteria-phage dynamics, and ultimately help identify key factors influencing the success of phage therapy.

Cancer-associated fibroblasts influence Wnt/PCP signaling in gastric cancer cells by cytoneme-based dissemination of ROR2.

Cancer-associated fibroblasts (CAFs) are a crucial component in the tumor microenvironment influencing cancer progression. Besides shaping the extracellular matrix, these fibroblasts provide signaling factors to facilitate tumor survival and alter tumor behavior. In gastric cancer, one crucial signaling pathway influencing invasion and metastasis is the Wnt/Planar Cell Polarity (PCP) signaling. The crucial PCP ligand in this context is WNT5A, which is produced by the CAFs, and gastric cancer cells react upon this signal by enhanced polarized migration. Why gastric cancer cells respond to this signal is still unclear, as their expression level for the central WNT5A receptor, ROR2, is very low. Here, we show that CAFs display long and branched filopodia that form an extensive, complex network engulfing gastric cancer cells, such as the gastric cancer cell line AGS. CAFs have a significantly higher expression level of ROR2 than normal gastric fibroblasts and AGS cells. By high-resolution imaging, we observe a direct transfer of fluorescently tagged ROR2 from CAF to AGS cells by signaling filopodia, known as cytonemes. Surprisingly, we find that the transferred ROR2 complexes can activate Wnt/JNK signaling in AGS cells. Consistently, blockage of ROR2 function in the CAFs leads to reduced paracrine Wnt/JNK signaling, cell polarization, and migration of the receiving AGS cells. Complementary, enhanced migration via paracrine ROR2 transfer was observed in a zebrafish in vivo model. These findings demonstrate a fresh role for cytoneme-mediated signaling in the tumor microenvironment. Cytonemes convey Wnt receptors from CAFs to gastric cancer cells, allowing them to respond to Wnt/PCP signals.

Phenotyping single-cell motility in microfluidic confinement.

The movement trajectories of organisms serve as dynamic read-outs of their behaviour and physiology. For microorganisms this can be difficult to resolve due to their small size and fast movement. Here, we devise a novel droplet microfluidics assay to encapsulate single micron-sized algae inside closed arenas, enabling ultralong high-speed tracking of the same cell. Comparing two model species - Chlamydomonas reinhardtii (freshwater, 2 cilia), and Pyramimonas octopus (marine, 8 cilia), we detail their highly-stereotyped yet contrasting swimming behaviours and environmental interactions. By measuring the rates and probabilities with which cells transition between a trio of motility states (smooth-forward swimming, quiescence, tumbling or excitable backward swimming), we reconstruct the control network that underlies this gait switching dynamics. A simplified model of cell-roaming in circular confinement reproduces the observed long-term behaviours and spatial fluxes, including novel boundary circulation behaviour. Finally, we establish an assay in which pairs of droplets are fused on demand, one containing a trapped cell with another containing a chemical that perturbs cellular excitability, to reveal how aneural microorganisms adapt their locomotor patterns in real-time.

High-Throughput Steady-State Enzyme Kinetics Measured in a Parallel Droplet Generation and Absorbance Detection Platform.

Microfluidic water-in-oil emulsion droplets are becoming a mainstay of experimental biology, where they replace the classical test tube. In most applications, such as ultrahigh-throughput directed evolution, the droplet content is identical for all compartmentalized assay reactions. When emulsion droplets are used for kinetics or other functional assays, though, concentration dependencies of initial rates that define Michaelis-Menten parameters are required. Droplet-on-demand systems satisfy this need, but extracting large amounts of data is challenging. Here, we introduce a multiplexed droplet absorbance detector, which─coupled to semi-automated droplet generation─forms a tubing-based droplet-on-demand system able to generate and extract quantitative datasets from defined concentration gradients across multiple series of droplets for multiple time points. The emergence of a product is detected by reading the absorbance of the droplet sets at multiple, adjustable time points by reversing the flow direction after each detection, so that the droplets pass a line scan camera multiple times. Detection multiplexing allows absorbance values at 12 distinct positions to be measured, and enzyme kinetics are recorded for label-free concentration gradients that are composed of about 60 droplets each, covering as many concentrations. With a throughput of around 8640 data points per hour, a 10-fold improvement compared to the previously reported single point detection method is achieved. In a single experiment, 12 full datasets of high-resolution and high-accuracy Michaelis-Menten kinetics were determined to demonstrate the potential for enzyme characterization for glycosidase substrates covering a range in enzymatic hydrolysis of 7 orders of magnitude in kcat/KM. The straightforward setup, high throughput, excellent data quality, and wide dynamic range that allows coverage of diverse activities suggest that this system may serve as a miniaturized spectrophotometer for detailed analysis of clones emerging from large-scale combinatorial experiments.

Materials and methods for droplet microfluidic device fabrication.

Since the first reports two decades ago, droplet-based systems have emerged as a compelling tool for microbiological and (bio)chemical science, with droplet flow providing multiple advantages over standard single-phase microfluidics such as removal of Taylor dispersion, enhanced mixing, isolation of droplet contents from surfaces, and the ability to contain and address individual cells or biomolecules. Typically, a droplet microfluidic device is designed to produce droplets with well-defined sizes and compositions that flow through the device without interacting with channel walls. Successful droplet flow is fundamentally dependent on the microfluidic device - not only its geometry but moreover how the channel surfaces interact with the fluids. Here we summarise the materials and fabrication techniques required to make microfluidic devices that deliver controlled uniform droplet flow, looking not just at physical fabrication methods, but moreover how to select and modify surfaces to yield the required surface/fluid interactions. We describe the various materials, surface modification techniques, and channel geometry approaches that can be used, and give examples of the decision process when determining which material or method to use by describing the design process for five different devices with applications ranging from field-deployable chemical analysers to water-in-water droplet creation. Finally we consider how droplet microfluidic device fabrication is changing and will change in the future, and what challenges remain to be addressed in the field.

Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats.

Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by -2.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended ß-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation.

Functional Phenotype Flow Cytometry: On Chip Sorting of Individual Cells According to Responses to Stimuli.

The ability to effectively separate and isolate biological cells into specific and well-defined subpopulations is crucial for the advancement of our understanding of cellular heterogeneity and its relevance to living systems. Here is described the development of the functional phenotype flow cytometer (FPFC), a new device designed to separate cells on the basis of their in situ real-time phenotypic responses to stimuli. The FPFC performs a cascade of cell processing steps on a microfluidic platform: introduces biological cells one at a time into a solution of a biological reagent that acts as a stimulus, incubates the cells with the stimulus solution in a flow, and sorts the cells into subpopulations according to their phenotypic responses to the provided stimulus. The presented implementation of the FPFC uses intracellular fluorescence as a readout, incubates cells for 75 s, and operates at a throughput of up to 4 cells min-1 -resulting in the profiling and sorting of hundreds of cells within a few hours. The design and operation of the FPFC are validated by sorting cells from the human Burkitt's lymphoma cancerous cell line Ramos on the basis of their response to activation of the B cell antigen receptor (BCR) by a targeted monoclonal antibody.

Deep learning guided image-based droplet sorting for on-demand selection and analysis of single cells and 3D cell cultures.

Uncovering the heterogeneity of cellular populations and multicellular constructs is a long-standing goal in fields ranging from antimicrobial resistance to cancer research. Emerging technology platforms such as droplet microfluidics hold the promise to decipher such heterogeneities at ultra-high-throughput. However, there is a lack of methods able to rapidly identify and isolate single cells or 3D cell cultures. Here we demonstrate that deep neural networks can accurately classify single droplet images in real-time based on the presence and number of micro-objects including single mammalian cells and multicellular spheroids. This approach also enables the identification of specific objects within mixtures of objects of different types and sizes. The training sets for the neural networks consisted of a few hundred images manually picked and augmented to up to thousands of images per training class. Training required less than 10 minutes using a single GPU, and yielded accuracies of over 90% for single mammalian cell identification. Crucially, the same model could be used to classify different types of objects such as polystyrene spheres, polyacrylamide beads and MCF-7 cells. We applied the developed method for the selection of 3D cell cultures generated with Hek293FT cells encapsulated in agarose gel beads, highlighting the potential of the technology for the selection of objects with a high diversity of visual appearances. The real-time sorting of single droplets was in-line with droplet generation and occurred at rates up to 40 per second independently of image size up to 480 × 480 pixels. The presented microfluidic device also enabled storage of sorted droplets to allow for downstream analyses.

Controlled Oil/Water Partitioning of Hydrophobic Substrates Extending the Bioanalytical Applications of Droplet-Based Microfluidics.

Functional annotation of novel proteins lags behind the number of sequences discovered by the next-generation sequencing. The throughput of conventional testing methods is far too low compared to sequencing; thus, experimental alternatives are needed. Microfluidics offer high throughput and reduced sample consumption as a tool to keep up with a sequence-based exploration of protein diversity. The most promising droplet-based systems have a significant limitation: leakage of hydrophobic compounds from water compartments to the carrier prevents their use with hydrophilic reagents. Here, we present a novel approach of substrate delivery into microfluidic droplets and apply it to high-throughput functional characterization of enzymes that convert hydrophobic substrates. Substrate delivery is based on the partitioning of hydrophobic chemicals between the oil and water phases. We applied a controlled distribution of 27 hydrophobic haloalkanes from oil to reaction water droplets to perform substrate specificity screening of eight model enzymes from the haloalkane dehalogenase family. This droplet-on-demand microfluidic system reduces the reaction volume 65 000-times and increases the analysis speed almost 100-fold compared to the classical test tube assay. Additionally, the microfluidic setup enables a convenient analysis of dependences of activity on the temperature in a range of 5 to 90 °C for a set of mesophilic and hyperstable enzyme variants. A high correlation between the microfluidic and test tube data supports the approach robustness. The precision is coupled to a considerable throughput of >20 000 reactions per day and will be especially useful for extending the scope of microfluidic applications for high-throughput analysis of reactions including compounds with limited water solubility.

Long-Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation.

Developmental cell biology requires technologies in which the fate of single cells is followed over extended time periods, to monitor and understand the processes of self-renewal, differentiation, and reprogramming. A workflow is presented, in which single cells are encapsulated into droplets (Ø: 80 µm, volume: ≈270 pL) and the droplet compartment is later converted to a hydrogel bead. After on-chip de-emulsification by electrocoalescence, these 3D scaffolds are subsequently arrayed on a chip for long-term perfusion culture to facilitate continuous cell imaging over 68 h. Here, the response of murine embryonic stem cells to different growth media, 2i and N2B27, is studied, showing that the exit from pluripotency can be monitored by fluorescence time-lapse microscopy, by immunostaining and by reverse-transcription and quantitative PCR (RT-qPCR). The defined 3D environment emulates the natural context of cell growth (e.g., in tissue) and enables the study of cell development in various matrices. The large scale of cell cultivation (in 2000 beads in parallel) may reveal infrequent events that remain undetected in lower throughput or ensemble studies. This platform will help to gain qualitative and quantitative mechanistic insight into the role of external factors on cell behavior.

Ultrahigh-Throughput Screening of Single-Cell Lysates for Directed Evolution and Functional Metagenomics.

The success of ultrahigh-throughput screening experiments in directed evolution or functional metagenomics strongly depends on the availability of efficient technologies for the quantitative testing of a large number of variants. With advanced robotics, libraries of up to 105 clones can be screened per day as colonies on agar plates or cell lysates in microwell plates, albeit at high cost of capital, manpower and consumables. These cost considerations and the general need for high-throughput make miniaturization of assay volumes attractive. To provide a general solution to maintain genotype-phenotype linkage, biochemical assays have been compartmentalized into water-in-oil droplets. This chapter presents a microfluidic workflow that translates a frequently used screening procedure consisting of cytoplasmic/periplasmic protein expression and cell lysis to the single cell level in water-in-oil droplet compartments. These droplets are sorted based on reaction progress by fluorescence measurements at the picoliter scale.

Exploring sequence space in search of functional enzymes using microfluidic droplets.

Screening of enzyme mutants in monodisperse picoliter compartments, generated at kilohertz speed in microfluidic devices, is coming of age. After a decade of proof-of-principle experiments, workflows have emerged that combine existing microfluidic modules to assay reaction progress quantitatively and yield improved enzymes. Recent examples of the screening of libraries of randomised proteins and from metagenomic sources suggest that this approach is not only faster and cheaper, but solves problems beyond the feasibility scope of current methodologies. The establishment of new assays in this format - so far covering hydrolases, aldolases, polymerases and dehydrogenases - will enable the exploration of sequence space for new catalysts of natural and non-natural chemical transformations.

Quantitative Affinity Determination by Fluorescence Anisotropy Measurements of Individual Nanoliter Droplets.

Fluorescence anisotropy measurements of reagents compartmentalized into individual nanoliter droplets are shown to yield high-resolution binding curves from which precise dissociation constants (Kd) for protein-peptide interactions can be inferred. With the current platform, four titrations can be obtained per minute (based on ∼100 data points each), with stoichiometries spanning more than 2 orders of magnitude and requiring only tens of microliters of reagents. In addition to affinity measurements with purified components, Kd values for unpurified proteins in crude cell lysates can be obtained without prior knowledge of the concentration of the expressed protein, so that protein purification can be avoided. Finally, we show how a competition assay can be set up to perform focused library screens, so that compound labeling is not required anymore. These data demonstrate the utility of droplet compartments for the quantitative characterization of biomolecular interactions and establish fluorescence anisotropy imaging as a quantitative technique in a miniaturized droplet format, which is shown to be as reliable as its macroscopic test tube equivalent.

Ultrahigh-throughput-directed enzyme evolution by absorbance-activated droplet sorting (AADS).

Ultrahigh-throughput screening, in which members of enzyme libraries compartmentalized in water-in-oil emulsion droplets are assayed, has emerged as a powerful format for directed evolution and functional metagenomics but is currently limited to fluorescence readouts. Here we describe a highly efficient microfluidic absorbance-activated droplet sorter (AADS) that extends the range of assays amenable to this approach. Using this module, microdroplets can be sorted based on absorbance readout at rates of up to 300 droplets per second (i.e., >1 million droplets per hour). To validate this device, we implemented a miniaturized coupled assay for NAD+-dependent amino acid dehydrogenases. The detection limit (10 µM in a coupled assay producing a formazan dye) enables accurate kinetic readouts sensitive enough to detect a minimum of 1,300 turnovers per enzyme molecule, expressed in a single cell, and released by lysis within a droplet. Sorting experiments showed that the AADS successfully enriched active variants up to 2,800-fold from an overwhelming majority of inactive ones at ∼100 Hz. To demonstrate the utility of this module for protein engineering, two rounds of directed evolution were performed to improve the activity of phenylalanine dehydrogenase toward its native substrate. Fourteen hits showed increased activity (improved >4.5-fold in lysate; kcat increased >2.7-fold), soluble protein expression levels (up 60%), and thermostability (Tm, 12 °C higher). The AADS module makes the most widely used optical detection format amenable to screens of unprecedented size, paving the way for the implementation of chromogenic assays in droplet microfluidics workflows.

Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics.

Unculturable bacterial communities provide a rich source of biocatalysts, but their experimental discovery by functional metagenomics is difficult, because the odds are stacked against the experimentor. Here we demonstrate functional screening of a million-membered metagenomic library in microfluidic picolitre droplet compartments. Using bait substrates, new hydrolases for sulfate monoesters and phosphotriesters were identified, mostly based on promiscuous activities presumed not to be under selection pressure. Spanning three protein superfamilies, these break new ground in sequence space: promiscuity now connects enzymes with only distantly related sequences. Most hits could not have been predicted by sequence analysis, because the desired activities have never been ascribed to similar sequences, showing how this approach complements bioinformatic harvesting of metagenomic sequencing data. Functional screening of a library of unprecedented size with excellent assay sensitivity has been instrumental in identifying rare genes constituting catalytically versatile hubs in sequence space as potential starting points for the acquisition of new functions.

A method to quantify FRET stoichiometry with phasor plot analysis and acceptor lifetime ingrowth.

FRET is widely used for the study of protein-protein interactions in biological samples. However, it is difficult to quantify both the FRET efficiency (E) and the affinity (Kd) of the molecular interaction from intermolecular FRET signals in samples of unknown stoichiometry. Here, we present a method for the simultaneous quantification of the complete set of interaction parameters, including fractions of bound donors and acceptors, local protein concentrations, and dissociation constants, in each image pixel. The method makes use of fluorescence lifetime information from both donor and acceptor molecules and takes advantage of the linear properties of the phasor plot approach. We demonstrate the capability of our method in vitro in a microfluidic device and also in cells, via the determination of the binding affinity between tagged versions of glutathione and glutathione S-transferase, and via the determination of competitor concentration. The potential of the method is explored with simulations.

Interfacing microwells with nanoliter compartments: a sampler generating high-resolution concentration gradients for quantitative biochemical analyses in droplets.

Analysis of concentration dependencies is key to the quantitative understanding of biological and chemical systems. In experimental tests involving concentration gradients such as inhibitor library screening, the number of data points and the ratio between the stock volume and the volume required in each test determine the quality and efficiency of the information gained. Titerplate assays are currently the most widely used format, even though they require microlitre volumes. Compartmentalization of reactions in pico- to nanoliter water-in-oil droplets in microfluidic devices provides a solution for massive volume reduction. This work addresses the challenge of producing microfluidic-based concentration gradients in a way that every droplet represents one unique reagent combination. We present a simple microcapillary technique able to generate such series of monodisperse water-in-oil droplets (with a frequency of up to 10 Hz) from a sample presented in an open well (e.g., a titerplate). Time-dependent variation of the well content results in microdroplets that represent time capsules of the composition of the source well. By preserving the spatial encoding of the droplets in tubing, each reactor is assigned an accurate concentration value. We used this approach to record kinetic time courses of the haloalkane dehalogenase DbjA and analyzed 150 combinations of enzyme/substrate/inhibitor in less than 5 min, resulting in conclusive Michaelis-Menten and inhibition curves. Avoiding chips and merely requiring two pumps, a magnetic plate with a stirrer, tubing, and a pipet tip, this easy-to-use device rivals the output of much more expensive liquid handling systems using a fraction (∼100-fold less) of the reagents consumed in microwell format.

A fully unsupervised compartment-on-demand platform for precise nanoliter assays of time-dependent steady-state enzyme kinetics and inhibition.

The ability to miniaturize biochemical assays in water-in-oil emulsion droplets allows a massive scale-down of reaction volumes, so that high-throughput experimentation can be performed more economically and more efficiently. Generating such droplets in compartment-on-demand (COD) platforms is the basis for rapid, automated screening of chemical and biological libraries with minimal volume consumption. Herein, we describe the implementation of such a COD platform to perform high precision nanoliter assays. The coupling of a COD platform to a droplet absorbance detection set-up results in a fully automated analytical system. Michaelis-Menten parameters of 4-nitrophenyl glucopyranoside hydrolysis by sweet almond ß-glucosidase can be generated based on 24 time-courses taken at different substrate concentrations with a total volume consumption of only 1.4 µL. Importantly, kinetic parameters can be derived in a fully unsupervised manner within 20 min: droplet production (5 min), initial reading of the droplet sequence (5 min), and droplet fusion to initiate the reaction and read-out over time (10 min). Similarly, the inhibition of the enzymatic reaction by conduritol B epoxide and 1-deoxynojirimycin was measured, and Ki values were determined. In both cases, the kinetic parameters obtained in droplets were identical within error to values obtained in titer plates, despite a >10(4)-fold volume reduction, from micro- to nanoliters.