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

Directed evolution and enzymatic biofuel cells,study of CotA laccase and its optimization by directed evolution using droplet based microfluidic

Les biopiles enzymatiques ont vu le jour pour développer des sources miniatures d'électricité renouvelable. Cette technologie naissante est cependant encore limitée en termes de puissance ou de durée de vie. Bien qu encore peu employée, une stratégie pourEnzymatic biofuel cells have been recently developed to create miniature renewable electricity sources. However, this new technology is still limited in terms of power and lifetime compared to classical fuel cells. Although it has been rarely used yet, o

Évolution dirigée et biopile enzymatique (étude de la laccase CotA et optimisation par évolution dirigée en microfluidique digitale)

Les biopiles enzymatiques ont vu le jour pour développer des sources miniatures d électricité renouvelable. Cette technologie naissante est cependant encore limitée en termes de puissance ou de durée de vie. Bien qu encore peu employée, une stratégie pour améliorer ces performances consiste à optimiser les propriétés catalytiques ou de stabilité des enzymes. Ces travaux de doctorat décrivent le développement d une plateforme de microfluidique digitale permettant l évolution dirigée de la laccase CotA de Bacillus subtilis pour une utilisation dans une biopile enzymatique. Ces travaux démontrent la possibilité d utiliser une enzyme extrêmophile au sein d une biopile enzymatique. L efficacité de CotA en tant que biocatalyseur de la réduction de l O2 a été évaluée pour la première fois en développant des biocathodes ou des biopiles Glucose/O2 complètes. Une plateforme microfluidique modèle de criblage à très haut débit applicable à l évolution dirigée de la laccase CotA a également été mise au point et validée. La plateforme permet l encapsulation de cellules E. coli exprimant la protéine dans des microgouttelettes aqueuses de quelques picolitres, l incubation des microgouttelettes, l ajout du substrat par picoinjection puis la détection et le tri de l activité enzymatique de CotA à très haut débit (1 million de clones en seulement 4 heures). La plateforme est directement applicable au criblage de banques de mutants et les variants optimisés sélectionnés devraient mener à la création d une nouvelle génération de biopiles plus efficaces. Cette plateforme universelle de criblage constitue un outil à la fois versatile et puissant pour l évolution dirigée des protéines.Enzymatic biofuel cells have been recently developed to create miniature renewable electricity sources. However, this new technology is still limited in terms of power and lifetime compared to classical fuel cells. Although it has been rarely used yet, one strategy to improve these performances is to optimize the catalytic and stability properties of the enzymes. This PhD work describes the development of a droplet-based microfluidic platform for thr directed evolution of CotA laccase from Bacillus subtilis for enzymatic biofuel cells application. This work demonstrates the possibility of using an extremophilic enzyme inside an enzymatique biofuel cell. The efficiency of CotA as a biocatalyst for O2 reduction has been evaluated for the first time developing biocathodes or complete Glucose/O2 biofuel cells. A droplet-based microfluidic high-throughput screening platform for CotA directed evolution has also been developed and validated. This platform allows the encapsulation of E. coli cells expressing the protein in aqueous droplets of few picoliters, the incubation of droplets, the addition of the substrate using picoinjection and then the detection and the sorting of CotA enzymatic activity using very high-throughput (1 million clones in only 4 hours). The platform can be directly used for the screening of mutant libraries. Optimized selected mutants would lead to the creation of a new and more efficient generation of enzymatic biofuel cells. This universal droplet-based microfluidic screening platform is a very powerful tool for directed evolution of proteins.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Hybrid bidentate phosphoramidite ligans in asymmetric catalysis

International audienc

Rational design of a high-throughput droplet sorter

a 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

The Thermophilic CotA Laccase from Bacillus subtilis: Bioelectrocatalytic Evaluation of O2 Reduction in the Direct and Mediated Electron Transfer Regime

International audienc

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

Immobilization of CotA, an extremophilic laccase from Bacillus subtilis, on glassy carbon electrodes for biofuel cell applications

International audienc

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.[br/] Results : Five hydrolytic genes from Aspergillus niger genome were chosen and the corresponding five Yarrowia lipolytica producing strains were constructed. Each enzyme (endo-β-1,4-xylanase B and C; 1,4-β-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-β-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).[br/] 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 (10(5) 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

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 at400 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 DCotA) 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....