Publications

Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100–1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450–900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility.

https://doi.org/10.1038/s41587-020-0466-7

Adjustment of plasmid copy number resulting from the balance between positive and negative impacts
of borne synthetic genes, plays a critical role in the global efficiency of multistep metabolic engineering.
Differential expression of co-expressed engineered genes is frequently observed depending on growth
phases, metabolic status and triggered adjustments of plasmid copy numbers, constituting a dynamic
process contributing to minimize global engineering burden. A yeast model involving plasmid based
expression of phosphoribulokinase (PRKp), a key enzyme for the reconstruction of synthetic Calvin cycle,
was designed to gain further insights into such a mechanism. A conditional PRK expression cassette was
cloned either onto a low (ARS-CEN based) or a high (2-micron origin based) copy number plasmid using
complementation of a trp1 genomic mutation as constant positive selection. Evolution of plasmid copy
numbers, PRKp expressions, and cell growth rates were dynamically monitored following gene derepression
through external doxycycline concentration shifts. In the absence of RubisCO encoding
gene permitting metabolic recycling, PRKp expression that led to depletion of ribulose phosphate, a
critical metabolite for aromatic amino-acids biosynthesis, and accumulation of the dead-end diphosphate
product contribute to toxicity. Triggered copy number adjustment was found to be a dynamic
process depending both on plasmid types and levels of PRK induction. With the ARS-CEN plasmid, cell
growth was abruptly affected only when level PRKp expression exceeded a threshold value. In contrast, a
proportional relationship was observed with the 2-micron plasmid consistent with large copy number
adjustments. Micro-compartment partitioning of bulk cultures by embedding individual cells into inverse
culture medium/oil droplets, revealed the presence of slow and fast growing subpopulations that
differ in relative proportions for low and high copy number plasmids.

https://doi.org/10.1016/j.resmic.2018.06.002

Studies of the dynamics of the antibody-mediated immune response have been hampered by the absence of quantitative, high-throughput systems to analyze individual antibody-secreting cells. Here we describe a simple microfluidic system, DropMap, in which single cells are compartmentalized in tens of thousands of 40-pL droplets and analyzed in two-dimensional droplet arrays using a fluorescence relocation-based immunoassay. Using DropMap, we characterized antibody-secreting cells in mice immunized with tetanus toxoid (TT) over a 7-week protocol, simultaneously analyzing the secretion rate and affinity of IgG from over 0.5 million individual cells enriched from spleen and bone marrow. Immunization resulted in dramatic increases in the range of both single-cell secretion rates and affinities, which spanned at maximum 3 and 4 logs, respectively. We observed differences over time in dynamics of secretion rate and affinity within and between anatomical compartments. This system will not only enable immune monitoring and optimization of immunization and vaccination protocols but also potentiate antibody screening

doi:10.1038/nbt.3964

access to a view-only version  http://rdcu.be/vKeI

We present the principle of a fast magnetic field enhanced colloidal agglutination assay, which is based on the acceleration of the recognition rate between ligands and receptors induced by magnetic forces. By applying a homogeneous magnetic field of 20 mT for only 7 s, we detect CRP (C-reactive protein) in human serum at a concentration as low as 1 pM for a total cycle time of about 1 min in a prototype analyzer. Such a short measurement time does not impair the performances of the assay when compared to longer experiments. The concentration range dynamic is shown to cover 3 orders of magnitude. An analytical model of agglutination is also successfully fitting our data obtained with a short magnetic pulse.

Microbiology has continuously pushed efforts towards understanding microbial diversity. Technologies and methods have also evolved, from plating, and use of mi- croscopes and cytometers, towards micro-well handling robots and, finally, fluidic- based devices. The aim of this review was to bring microbiologists attention to the outstanding analytical and handling power of millifluidic droplet technologies for analysing and sorting phenotypic diversity in the microbial world. This new format overcomes many limitations of previous approaches. It provides outstanding repro- ducible growth conditions over droplet reservoirs allowing unprecedented sensitive read-out over thousands of colonies over time. The confinement of the millifluidic train within tubes and the implementation of a three phases format excludes any con- tamination issues. The automation and handling of reservoir droplets is inherently facilitated. We show as a proof of principle the efficiency of capturing phenotypic diversity within a bacterial sample submitted to a sub-minimum inhibitory con- centration of antibiotic. The precision offered by the millifluidic format allows the detection of a variety of resistance strategies that compete and coexist. The review finally explores the potential of this approach to address new challenges such as community-based growth of multiple-strain systems.

To address possible cell-to-cell heterogeneity in growth dynamics of isogenic cell popula- tions of Chlamydomonas reinhardtii, we developed a millifluidic drop-based device that not only allows the analysis of populations grown from single cells over periods of a week, but is also able to sort and collect drops of interest, containing viable and healthy cells, which can be used for further experimentation. In this study, we used isogenic algal cells that were first synchronized in mixotrophic growth conditions. We show that these synchronized cells, when placed in droplets and kept in mixotrophic growth conditions, exhibit mostly homoge- neous growth statistics, but with two distinct subpopulations: a major population with a short doubling-time (fast-growers) and a significant subpopulation of slowly dividing cells (slow- growers). These observations suggest that algal cells from an isogenic population may be present in either of two states, a state of restricted division and a state of active division. When isogenic cells were allowed to propagate for about 1000 generations on solid agar plates, they displayed an increased heterogeneity in their growth dynamics. Although we could still identify the original populations of slow- and fast-growers, drops inoculated with a single progenitor cell now displayed a wider diversity of doubling-times. Moreover, popula- tions dividing with the same growth-rate often reached different cell numbers in stationary phase, suggesting that the progenitor cells differed in the number of cell divisions they could undertake. We discuss possible explanations for these cell-to-cell heterogeneities in growth dynamics, such as mutations, differential aging or stochastic variations in metabolites and macromolecules yielding molecular switches, in the light of single-cell heterogeneities that have been reported among isogenic populations of other eu- and prokaryotes.

En 10 ans le coût du séquençage d’un génome a été divisé par 10 000. Alors qu’il fallait trois ans pour séquencer le génome humain il ne faut plus que 10 jours avec les séquenceurs de dernière génération. Ces progrès technologiques sur la « lecture » du code génétique ont des répercussions sur de nombreux domaines de la biologie.