Coacervate Droplets for Synthetic Cells.

The design and construction of synthetic cells - human-made microcompartments that mimic features of living cells - have experienced a real boom in the past decade. While many efforts have been geared toward assembling membrane-bounded compartments, coacervate droplets produced by liquid-liquid phase separation have emerged as an alternative membrane-free compartmentalization paradigm. Here, the dual role of coacervate droplets in synthetic cell research is discussed: encapsulated within membrane-enclosed compartments, coacervates act as surrogates of membraneless organelles ubiquitously found in living cells; alternatively, they can be viewed as crowded cytosol-like chassis for constructing integrated synthetic cells. After introducing key concepts of coacervation and illustrating the chemical diversity of coacervate systems, their physicochemical properties and resulting bioinspired functions are emphasized. Moving from suspensions of free floating coacervates, the two nascent roles of these droplets in synthetic cell research are highlighted: organelle-like modules and cytosol-like templates. Building the discussion on recent studies from the literature, the potential of coacervate droplets to assemble integrated synthetic cells capable of multiple life-inspired functions is showcased. Future challenges that are still to be tackled in the field are finally discussed.

Directed Evolution in Drops: Molecular Aspects and Applications.

The process of optimizing the properties of biological molecules is paramount for many industrial and medical applications. Directed evolution is a powerful technique for modifying and improving biomolecules such as proteins or nucleic acids (DNA or RNA). Mimicking the mechanism of natural evolution, one can enhance a desired property by applying a suitable selection pressure and sorting improved variants. Droplet-based microfluidic systems offer a high-throughput solution to this approach by helping to overcome the limiting screening steps and allowing the analysis of variants within increasingly complex libraries. Here, we review cases where successful evolution of biomolecules was achieved using droplet-based microfluidics, focusing on the molecular processes involved and the incorporation of microfluidics to the workflow. We highlight the advantages and limitations of these microfluidic systems compared to low-throughput methods and show how the integration of these systems into directed evolution workflows can open new avenues to discover or improve biomolecules according to user-defined conditions.

Confining Trypanosoma brucei in emulsion droplets reveals population variabilities in division rates and improves in vitro cultivation.

Trypanosome parasites are infecting mammals in Sub-Saharan Africa and are transmitted between hosts through bites of the tsetse fly. The transmission from the insect vector to the mammal host causes a number of metabolic and physiological changes. A fraction of the population continuously adapt to the immune system of the host, indicating heterogeneity at the population level. Yet, the cell to cell variability in populations is mostly unknown. We develop here an analytical method for quantitative measurements at the single cell level based on encapsulation and cultivation of single-cell Trypanosoma brucei in emulsion droplets. We first show that mammalian stage trypanosomes survive for several hours to days in droplets, with an influence of droplet size on both survival and growth. We unravel various growth patterns within a population and find that droplet cultivation of trypanosomes results in 10-fold higher cell densities of the highest dividing cell variants compared to standard cultivation techniques. Some variants reach final cell titers in droplets closer to what is observed in nature than standard culture, of practical interest for cell production. Droplet microfluidics is therefore a promising tool for trypanosome cultivation and analysis with further potential for high-throughput single cell trypanosome analysis.

Droplet-Based Microfluidic High-Throughput Screening of Enzyme Mutant Libraries Secreted by Yarrowia lipolytica.

Yarrowia lipolytica has emerged as an attractive solution for screening enzyme activities thanks to the numerous tools available for heterologous protein production and its strong secretory ability. Nowadays, activity screening for improved enzymes mostly relies on the evaluation of independent clones in microtiter plates. However, even with highly robotized screening facilities, the relatively low throughput and high cost of the technology do not enable the screening of large diversities, which significantly reduce the probability of isolating improved variants. Droplet-based microfluidics is an emerging technology that allows the high-throughput and individual picoliter droplets manipulation and sorting based on enzymatic substrate fluorescence. This technology is an attractive alternative to microtiter plate screenings with higher throughputs and drastic reduction of working volume and cost.Here, we present a droplet-based microfluidic platform for the screening of libraries expressed in the yeast Y. lipolytica, from the generation of a random mutagenesis library of a heterologous enzyme and its expression in Y. lipolytica to the droplet-based microfluidic procedures composed of cell encapsulation and growth and activity screening or sorting of improved clones.

Variable inter and intraspecies alkaline phosphatase activity within single cells of revived dinoflagellates.

Adaptation of cell populations to environmental changes is mediated by phenotypic variability at the single-cell level. Enzyme activity is a key factor in cell phenotype and the expression of the alkaline phosphatase activity (APA) is a fundamental phytoplankton strategy for maintaining growth under phosphate-limited conditions. Our aim was to compare the APA among cells and species revived from sediments of the Bay of Brest (Brittany, France), corresponding to a pre-eutrophication period (1940's) and a beginning of a post-eutrophication period (1990's) during which phosphate concentrations have undergone substantial variations. Both toxic marine dinoflagellate Alexandrium minutum and the non-toxic dinoflagellate Scrippsiella acuminata were revived from ancient sediments. Using microfluidics, we measured the kinetics of APA at the single-cell level. Our results indicate that all S. acuminata strains had significantly higher APA than A. minutum strains. For both species, the APA in the 1990's decade was significantly lower than in the 1940's. For the first time, our results reveal both inter and intraspecific variabilities of dinoflagellate APA and suggest that, at a half-century timescale, two different species of dinoflagellate may have undergone similar adaptative evolution to face environmental changes and acquire ecological advantages.

Light-powered CO2 fixation in a chloroplast mimic with natural and synthetic parts.

Nature integrates complex biosynthetic and energy-converting tasks within compartments such as chloroplasts and mitochondria. Chloroplasts convert light into chemical energy, driving carbon dioxide fixation. We used microfluidics to develop a chloroplast mimic by encapsulating and operating photosynthetic membranes in cell-sized droplets. These droplets can be energized by light to power enzymes or enzyme cascades and analyzed for their catalytic properties in multiplex and real time. We demonstrate how these microdroplets can be programmed and controlled by adjusting internal compositions and by using light as an external trigger. We showcase the capability of our platform by integrating the crotonyl-coenzyme A (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle, a synthetic network for carbon dioxide conversion, to create an artificial photosynthetic system that interfaces the natural and the synthetic biological worlds.

Bacterial Expression Systems for Enzymatic Activity in Droplet-Based Microfluidics.

Functional screenings in droplet-based microfluidics require the analysis of various types of activities of individual cells. When screening for enzymatic activities, the link between the enzyme of interest and the information-baring molecule, the DNA, must be maintained to relate phenotypes to genotypes. This linkage is crucial in directed evolution experiments or for the screening of natural diversity. Micro-organisms are classically used to express enzymes from nucleic acid sequences. However, little information is available regarding the most suitable expression system for the sensitive detection of enzymatic activity at the single-cell level in droplet-based microfluidics. Here, we compare three different expression systems for l-asparaginase (l-asparagine amidohydrolase, EC 3.5.1.1), an enzyme of therapeutic interest that catalyzes the conversion of l-asparagine to l-aspartic acid and ammonia. We developed three expression vectors to produce and localize l-asparaginase (l-ASNase) in E. coli either in the cytoplasm, on the surface of the inner membrane (display), or in the periplasm. We show that the periplasmic expression is the most optimal strategy combining both a good yield and a good accessibility for the substrate without the need for lysing the cells. We suggest that periplasmic expression may provide a very efficient platform for screening applications at the single-cell level in microfluidics.

Rational design of a high-throughput droplet sorter.

The high-throughput selection of individual droplets is an essential function in droplet-based microfluidics. Fluorescence-activated droplet sorting is achieved using electric fields triggered at rates up to 30 kHz, providing the ultra-high throughput relevant in applications where large libraries of compounds or cells must be analyzed. To achieve such sorting frequencies, electrodes have to create an electric field distribution that generates maximal actuating forces on the droplet while limiting the induced droplet deformation and avoid disintegration. We propose a metric characterizing the performance of an electrode design relative to the theoretical optimum and analyze existing devices using full 3D simulations of the electric fields. By combining parameter optimization with numerical simulation we derive rational design guidelines and propose optimized electrode configurations. When tested experimentally, the optimized design show significantly better performance than the standard designs.

Microfluidic technology for plankton research.

Plankton produces numerous chemical compounds used in cosmetics and functional foods. They also play a key role in the carbon budget on the Earth. In a context of global change, it becomes important to understand the physiological response of these microorganisms to changing environmental conditions. Their adaptations and the response to specific environmental conditions are often restricted to a few active cells or individuals in large populations. Using analytical capabilities at the subnanoliter scale, microfluidic technology has also demonstrated a high potential in biological assays. Here, we review recent advances in microfluidic technologies to overcome the current challenges in high content analysis both at population and the single cell level.

Out-of-equilibrium microcompartments for the bottom-up integration of metabolic functions.

Self-sustained metabolic pathways in microcompartments are the corner-stone for living systems. From a technological viewpoint, such pathways are a mandatory prerequisite for the reliable design of artificial cells functioning out-of-equilibrium. Here we develop a microfluidic platform for the miniaturization and analysis of metabolic pathways in man-made microcompartments formed of water-in-oil droplets. In a modular approach, we integrate in the microcompartments a nicotinamide adenine dinucleotide (NAD)-dependent enzymatic reaction and a NAD-regeneration module as a minimal metabolism. We show that the microcompartments sustain a metabolically active state until the substrate is fully consumed. Reversibly, the external addition of the substrate reboots the metabolic activity of the microcompartments back to an active state. We therefore control the metabolic state of thousands of independent monodisperse microcompartments, a step of relevance for the construction of large populations of metabolically active artificial cells.

Preparation of Swellable Hydrogel-Containing Colloidosomes from Aqueous Two-Phase Pickering Emulsion Droplets.

The fabrication of stable colloidosomes derived from water-in-water Pickering-like emulsions are described that were produced by addition of fluorescent amine-modified polystyrene latex beads to an aqueous two-phase system consisting of dextran-enriched droplets dispersed in a PEG-enriched continuous phase. Addition of polyacrylic acid followed by carbodiimide-induced crosslinking with dextran produces hydrogelled droplets capable of reversible swelling and selective molecular uptake and exclusion. Colloidosomes produced specifically in all-water systems could offer new opportunities in microencapsulation and the bottom-up construction of synthetic protocells.

High-Content Screening of Plankton Alkaline Phosphatase Activity in Microfluidics.

One way for phytoplankton to survive orthophosphate depletion is to utilize dissolved organic phosphorus by expressing alkaline phosphatase. The actual methods to assay alkaline phosphate activity-either in bulk or as a presence/absence of enzyme activity-fail to provide information on individual living cells. In this context, we develop a new microfluidic method to compartmentalize cells in 0.5 nL water-in-oil droplets and measure alkaline phosphatase activity at the single-cell level. We use enzyme-labeled fluorescence (ELF), which is based on the hydrolysis of ELF-P substrate, to monitor in real time and at the single-cell level both qualitative and quantitative information on cell physiology (i.e., localization and number of active enzyme sites and alkaline phosphatase kinetics). We assay the alkaline phosphatase activity of Tetraselmis sp. as a function of the dissolved inorganic phosphorus concentration and show that the time scale of the kinetics spans 1 order of magnitude. The advantages of subnanoliter-scale compartmentalization in droplet-based microfluidics provide a precise characterization of a population with single-cell resolution. Our results highlight the key role of cell physiology to efficiently access dissolved organic phosphorus.

Catanionic Coacervate Droplets as a Surfactant-Based Membrane-Free Protocell Model.

We report on the formation of surfactant-based complex catanionic coacervate droplets in mixtures of decanoic acid and cetylpyridinium chloride or cetyltrimethylammonium bromide. We show that coacervation occurs over a broad range of composition, pH, and ionic strength. The catanionic coacervates consist of elongated micelles, sequester a wide range of solutes including water-soluble organic dyes, polysaccharides, proteins, enzymes, and DNA, and can be structurally stabilized by sodium alginate or gelatin-based hydrogelation. These results suggest that catanionic coacervates could be exploited as a novel surfactant-based membrane-free protocell model.

Droplet-based microfluidic high-throughput screening of heterologous enzymes secreted by the yeast Yarrowia lipolytica.

BACKGROUND: Droplet-based microfluidics is becoming an increasingly attractive alternative to microtiter plate techniques for enzymatic high-throughput screening (HTS), especially for exploring large diversities with lower time and cost footprint. In this case, the assayed enzyme has to be accessible to the substrate within the water-in-oil droplet by being ideally extracellular or displayed at the cell surface. However, most of the enzymes screened to date are expressed within the cytoplasm of Escherichia coli cells, which means that a lysis step must take place inside the droplets for enzyme activity to be assayed. Here, we take advantage of the excellent secretion abilities of the yeast Yarrowia lipolytica to describe a highly efficient expression system particularly suitable for the droplet-based microfluidic HTS. RESULTS: Five hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-ß-1,4-xylanase B and C; 1,4-ß-cellobiohydrolase A; endoglucanase A; aspartic protease) was successfully overexpressed and secreted in an active form in the crude supernatant. A droplet-based microfluidic HTS system was developed to (a) encapsulate single yeast cells; (b) grow yeast in droplets; (c) inject the relevant enzymatic substrate; (d) incubate droplets on chip; (e) detect enzymatic activity; and (f) sort droplets based on enzymatic activity. Combining this integrated microfluidic platform with gene expression in Y. lipolytica results in remarkably low variability in the enzymatic activity at the single cell level within a given monoclonal population (<5%). Xylanase, cellobiohydrolase and protease activities were successfully assayed using this system. We then used the system to screen for thermostable variants of endo-ß-1,4-xylanase C in error-prone PCR libraries. Variants displaying higher thermostable xylanase activities compared to the wild-type were isolated (up to 4.7-fold improvement). CONCLUSIONS: Yarrowia lipolytica was used to express fungal genes encoding hydrolytic enzymes of interest. We developed a successful droplet-based microfluidic platform for the high-throughput screening (105 strains/h) of Y. lipolytica based on enzyme secretion and activity. This approach provides highly efficient tools for the HTS of recombinant enzymatic activities. This should be extremely useful for discovering new biocatalysts via directed evolution or protein engineering approaches and should lead to major advances in microbial cell factory development.

High-throughput screening of filamentous fungi using nanoliter-range droplet-based microfluidics.

Filamentous fungi are an extremely important source of industrial enzymes because of their capacity to secrete large quantities of proteins. Currently, functional screening of fungi is associated with low throughput and high costs, which severely limits the discovery of novel enzymatic activities and better production strains. Here, we describe a nanoliter-range droplet-based microfluidic system specially adapted for the high-throughput sceening (HTS) of large filamentous fungi libraries for secreted enzyme activities. The platform allowed (i) compartmentalization of single spores in ~10 nl droplets, (ii) germination and mycelium growth and (iii) high-throughput sorting of fungi based on enzymatic activity. A 10(4) clone UV-mutated library of Aspergillus niger was screened based on α-amylase activity in just 90 minutes. Active clones were enriched 196-fold after a single round of microfluidic HTS. The platform is a powerful tool for the development of new production strains with low cost, space and time footprint and should bring enormous benefit for improving the viability of biotechnological processes.

Droplet-based microfluidics platform for ultra-high-throughput bioprospecting of cellulolytic microorganisms.

Discovery of microorganisms producing enzymes that can efficiently hydrolyze cellulosic biomass is of great importance for biofuel production. To date, however, only a miniscule fraction of natural biodiversity has been tested because of the relatively low throughput of screening systems and their limitation to screening only culturable microorganisms. Here, we describe an ultra-high-throughput droplet-based microfluidic system that allowed the screening of over 100,000 cells in less than 20 min. Uncultured bacteria from a wheat stubble field were screened directly by compartmentalization of single bacteria in 20 pl droplets containing a fluorogenic cellobiohydrolase substrate. Sorting of droplets based on cellobiohydrolase activity resulted in a bacterial population with 17- and 7-fold higher cellobiohydrolase and endogluconase activity, respectively, and very different taxonomic diversity than when selected for growth on medium containing starch and carboxymethylcellulose as carbon source.

CotA laccase: high-throughput manipulation and analysis of recombinant enzyme libraries expressed in E. coli using droplet-based microfluidics.

We present a high-throughput droplet-based microfluidic analysis/screening platform for directed evolution of CotA laccase: droplet-based microfluidic modules were combined to develop an efficient system that allows cell detection and sorting based on the enzymatic activity. This platform was run on two different operating modes: the "analysis" mode allowing the analysis of the enzymatic activity in droplets at very high rates (>1000 Hz) and the "screening" mode allowing sorting of active droplets at 400 Hz. The screening mode was validated for the directed evolution of the cytoplasmic CotA laccase from B. subtilis, a potential interesting thermophilic cathodic catalyst for biofuel cells. Single E. coli cells expressing either the active CotA laccase (E. coli CotA) or an inactive frameshifted variant (E. coli ΔCotA) were compartmentalized in aqueous droplets containing expression medium. After cell growth and protein expression within the droplets, a fluorogenic substrate was "picoinjected" in each droplet. Fluorescence-activated droplet sorting was then used to sort the droplets containing the desired activity and the corresponding cells were then recultivated and identified using colorimetric assays. We demonstrated that E. coli CotA cells were enriched 191-fold from a 1 : 9 initial ratio of E. coli CotA to E. coli ΔCotA cells (or 437-fold from a 1 : 99 initial ratio) using a sorting rate of 400 droplets per s. This system allows screening of 10(6) cells in only 4 h, compared to 11 days for screening using microtitre plate-based systems. Besides this low error rate sorting mode, the system can also be used at higher throughputs in "enrichment" screening mode to make an initial purification of a library before further steps of selection. Analysis mode, without sorting, was used to rapidly quantify the activity of a CotA library constructed using error-prone PCR. This mode allows analysis of 10(6) cells in only 1.5 h.