Publications

Droplet-based microfluidics, using water-in-oil emulsion droplets as microreactors, is becoming a widespread method for performing assays and especially in the cell biology field. Making a simple and highly portable system for creating emulsion droplets would help to continue the popularization of such a technique. Also, the ability to emulsify all the samples would strengthen this compartimenlization technique to handle samples with limited volume. Here, we propose a strategy of droplet formation that combines a classical flow-focusing microfluidic chip, which could be commercially available, with a standard laboratory adjustable micropipette. The micropipette is used as a negative pressure generator for controlling liquid flows. In that way, emulsification does neither require any electrical power supply nor a cumbersome device and functions with small liquid volumes. Droplet formation can be easily and safely performed in places with limited space, opening a wide range of applications especially in biological laboratory environments with higher level of safety regulations, i.e., BSL-3/4. Fortunately, the present methodology that involves small fluid volumes, and thus possible time dependent flow conditions, allows to minimize dead volume while keeping drops’ size homogeneous. A physical characterization of droplet production and a model that describes the emulsion features, in terms of drop size and size distribution, are proposed for rationalizing the performances of the micropipette-powered emulsification process.

The osmotic pressure of polyelectrolyte solutions as a function of concentration has been calculated by Monte Carlo simulations of a spherical cell model and by molecular dynamics simulations with periodic boundary conditions. The results for the coarse-grained polyelectrolyte model are in good agreement with experimental results for sodium polyacrylate and the cell model is validated by the bulk simulations. The cell model offers an alternative perspective on osmotic pressure and also forms a direct link to even simpler models in the form of the Poisson-Boltzmann approximation applied to cylindrical and spherical geometries. As a result, the non-monotonic behaviour of the osmotic coefficient seen in simulated salt-free solutions is shown not to rely on a transition between a dilute and semi-dilute regime, as is often suggested when the polyion is modelled as a linear flexible chain. The non-monotonic behaviour is better described as the combination of a finite-size effect and a double-layer effect. Parameters that represent the linear nature of the polyion, including an alternative to monomer concentration, make it possible to display a generalised behaviour of equivalent chains, at least at low concentrations. At high concentrations, local interactions become significant and the exact details of the model become important. The effects of added salt are also discussed and one conclusion is that the empirical additivity rule, treating the contributions from the polyelectrolyte and any salt separately, is a reasonable approximation, which justifies the study of salt-free solutions.

Some bacteria can act as catalysts to oxidize (or reduce) organic or inorganic matter with the potential of generating electrical current. Despite their high value for sustainable energy, organic compound production and bioremediation, a tool to probe the natural biodiversity and to select most efficient microbes is still lacking. Compartmentalized cell culture is an ideal strategy for achieving such a goal but the appropriate compartment allowing cell growth and electron exchange must be tailored. Here, we develop a conductive composite hydrogel made of a double network of alginate and carbon nanotubes. Homogeneous mixing of carbon nanotubes within the polyelectrolyte is obtained by a surfactant assisted dispersion followed by a desorption step for triggering electrical conductivity. Dripping the mixture in a gelling bath through simple extrusion or a double one allows the formation of either plain hydrogel beads or liquid core hydrogel capsules. The process is shown to be compatible with the bacterial culture (Geobacter sulfurreducens). Bacteria can indeed colonize the outer wall of plain beads or the inner wall of the conductive capsules’ shell that function as an anode from which electrons produced by the cells are collected.

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

Application of droplet-based microfluidics for the screening of microbial libraries is one of the important ongoing developments in functional genomics/metagenomics. In this article, we propose a new method that can be employed for high-throughput profiling of cell growth. It consists of light-driven labelling droplets that contain growing cells directly in a microfluidics observation chamber, followed by recovery of the labelled cells. This method is based on intracellular expression of green-to-red switchable fluorescent proteins. The proof of concept is established here for two commonly used biological models, E. coli and S. cerevisiae. Growth of cells in droplets was monitored under a microscope and, depending on the targeted phenotype, the fluorescence of selected droplets was switched from a “green” to a “red” state. Red fluorescent cells from labelled droplets were then successfully detected, sorted with the Fluorescence Activated Cell Sorting machine and recovered. Finally, the application of this method for different kind of screenings, in particular of metagenomic libraries, is discussed and this idea is validated by the analysis of a model mini-library.

https://doi.org/10.1016/j.mimet.2018.03.001