Publications

SEARCH

Laboratory :
Author :
Revue :
Year :

Polyadenylation of a functional mRNA controls gene expression in Escherichia coli
Joanny G, Le Derout J, Bréchemier-Baey D, Labas V, Vinh J, Régnier P, Hajnsdorf E.
Nucleic Acids Res. - 35(8) :2494-502 - PMID:17395638 - 2007
Although usually implicated in the stabilization of mRNAs in eukaryotes, polyadenylation was initially shown to destabilize RNA in bacteria. All the data are consistent with polyadenylation being part of a quality control process targeting folded RNA fragments and non-functional RNA molecules to degradation. We report here an example in Escherichia coli, where polyadenylation directly controls the level of expression of a gene by modulating the stability of a functional transcript. Inactivation of poly(A)polymerase I causes overexpression of glucosamine-6-phosphate synthase (GlmS) and both the accumulation and stabilization of the glmS transcript. Moreover, we show that the glmS mRNA results from the processing of the glmU-glmS cotranscript by RNase E. Interestingly, the glmU-glmS cotranscript and the mRNA fragment encoding GlmU only slightly accumulated in the absence of poly(A)polymerase, suggesting that the endonucleolytically generated glmS mRNA harbouring a 5' monophosphate and a 3' stable hairpin is highly susceptible to poly(A)-dependent degradation.
Signal enhancement in electronic detection of DNA hybridization
C. Gentil, G. Philippin, and U. Bockelmann
Phys. Rev. E - 75(1) :011926 - DOI:10.1103/PhysRevE.75.011926 - 2007
Electronic detection of the specific recognition between complementary DNA sequences is investigated. DNA probes are immobilized at different lateral positions on a Poly(L-lysine)-coated surface of an integrated silicon transistor array. Hybridization and field effect detection are done with the solid surface immersed in electrolyte solutions. Differential measurements are performed, where DNA hybridization leads to surface potential shifts between the transistors of the array. We experimentally show that these differential signals of hybridization can be enhanced significantly by changing the salt concentration between hybridization and detection.
Micropipette-powered droplet based microfluidics
Krzysztof Langer, Nicolas Bremond, Laurent Boitard, Jean Baudry, Jerome Bibette
Biomicrofluidics - 12 44106 - https://doi.org/10.1063/1.5037795 -
Droplet-based microfluidics, using water-in-oil emulsion droplets as micro-reactors, is
becoming a widespread method for performing assays and especially in the cell biol-
ogy 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 han-
dle samples with limited volume. Here, we propose a strategy of droplet formation
that combines a classical flow-focusing microfluidic chip, which could be commer-
cially available, with a standard laboratory adjustable micropipette. The micropipette
is used as a negative pressure generator for controlling liquid flows. In that way, emul-
sification 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 espe-
cially in biological laboratory environments with higher level of safety regulations,
i.e., BSL-3/4. Fortunately, the present methodology that involves small fluid vol-
umes, 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.
Published by AIP Publishing.
Micropipette-powered droplet based microfluidics
Krzysztof Langer, Nicolas Bremonda), Laurent Boitard, Jean Baudry, and Jérôme Bibette
Biomicrofluidics - 44106 - 10.1063/1.5037795 -
Droplet-based microfluidics, using water-in-oil emulsion droplets as micro-reactors, 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.
Rouzeau C, Dagkesamanskaya A, Langer K, Bibette J, Baudry J, Pompon D, Anton-Leberre V.
- 169(6) 335-342. - doi: 10.1016/j.resmic.2018.06.002. -
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 de-repression 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.
Dagkesamanskaya A, Langer K, Tauzin AS, Rouzeau C, Lestrade D, Potocki-Veronese G, Boitard L, Bibette J, Baudry J, Pompon D, Anton-Leberre V.
J Microbiol Methods. - 147 59-65 - doi: 10.1016/j.mimet.2018.03.001. -
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.

Copyright © 2018 Elsevier B.V. All rights reserved.
The mechanism of eccrine sweat pore plugging by aluminium salts using microfluidics combined with small angle X-ray scattering
Alice Bretagne, Franck Cotot, Mireille Arnaud-Roux, Michael Sztucki, Bernard Cabane and Jean-Baptiste Galey
Soft Matter - 13 3812-3821 - 10.1039/C6SM02510B -
Aluminium salts are widely used to control sweating for personal hygiene purposes. Their mechanism of action as antiperspirants was previously thought to be a superficial plugging of eccrine sweat pores by the aluminium hydroxide gel. Here we present a microfluidic T junction device that mimics sweat ducts, and is designed for the real time study of interactions between sweat and ACH (Aluminium Chloro Hydrate) under conditions that lead to plug formation. We used this device to image and measure the diffusion of aluminium polycationic species in sweat counter flow. We report the results of small angle X-ray scattering experiments performed to determine the structure and composition of the plug, using BSA (Bovine Serum Albumin) as a model of sweat proteins. Our results show that pore occlusion occurs as a result of the aggregation of sweat proteins by aluminium polycations. Mapping of the device shows that this aggregation is initiated in the T junction at the location where the flow of aluminium polycations joins the flow of BSA. The mechanism involves two stages: (1) a nucleation stage in which aggregates of protein and polycations bind to the wall of the sweat duct and form a tenuous membrane, which extends across the junction; (2) a growth stage in which this membrane collects proteins that are carried by hydrodynamic flow in the sweat channel and polycations that diffuse into this channel. These results could open up perspectives to find new antiperspirant agents with an improved efficacy.
Osmotic pressures of lysozyme solutions from gas-like to crystal states
Coralie Pasquier,ab Sylvie Beaufils,b Antoine Bouchoux, Sophie Rigault, Bernard Cabane, Mikael Lund, Valérie Lechevalier,a Cécile Le Floch-Fouéré,a Maryvonne Pasco,a Gilles Pabœuf,b Javier Pérezf and Stéphane Pezennec*a
Phys. Chem. - 18 28458-28465 - DOI: 10.1039/C6CP03867K -
We obtained osmotic pressure data of lysozyme solutions, describing their physical states over a wide concentration range, using osmotic stress for pressures between 0.05 bar and about 40 bar and volume fractions between 0.01 and 0.61. The osmotic pressure vs. volume fraction data consist of a dilute, gas-phase regime, a transition regime with a high-compressibility plateau, and a concentrated regime where the system is nearly incompressible. The first two regimes are shifted towards a higher protein volume fraction upon decreasing the strength or the range of electrostatic interactions. We describe this shift and the overall shape of the experimental data in these two regimes through a model accounting for a steric repulsion, a short-range van der Waals attraction and a screened electrostatic repulsion. The transition is caused by crystallization, as shown by small-angle X-ray scattering. We verified that our data points correspond to thermodynamic equilibria, and thus that they consist of the reference experimental counterpart of a thermodynamic equation of state.
Coarse-grained modeling of the intrinsically disordered protein Histatin 5 in solution: Monte Carlo simulations in combination with SAXS.
Cragnell C, Durand D, Cabane B, Skepö
Proteins - 84(6) 777-91 - doi: 10.1002/prot.25025. -
Monte Carlo simulations and coarse-grained modeling have been used to analyze Histatin 5, an unstructured short cationic salivary peptide known to have anticandidical properties. The calculated scattering functions have been compared with intensity curves and the distance distribution function P(r) obtained from small angle X-ray scattering (SAXS), at both high and low salt concentrations. The aim was to achieve a molecular understanding and a physico-chemical insight of the obtained SAXS results and to gain information of the conformational changes of Histatin 5 due to altering salt content, charge distribution, and net charge. From a modeling perspective, the accuracy of the electrostatic interactions are of special interest. The used coarse-grained model was based on the primitive model in which charged hard spheres differing in charge and in size represent the ionic particles, and the solvent only enters the model through its relative permittivity. The Hamiltonian of the model comprises three different contributions: (i) excluded volumes, (ii) electrostatic, and (iii) van der Waals interactions. Even though the model can be considered as gross omitting all atomistic details, a great correspondence is obtained with the experimental results. Proteins 2016; 84:777-791.
Traffic collision during the breakup of an aqueous viscous compound jet
Hugo Doméjean, Jérôme Bibette, and Nicolas Bremond
Phys. Rev. Fluids - 1 63903 - https://doi.org/10.1103/PhysRevFluids.1.063903 -
Liquid jets ultimately break up into droplets through an instability driven by surface tension. For highly viscous liquids, drops are connected by cylindrical liquid filaments whose radii linearly decrease with time, thus forming drops on a string structure. For a jet composed of two aqueous phases made in air by coaxial extrusion, we observe that, for moderate Weber and capillary numbers, drops slow down with different velocities, leading to drop coalescence. The origin of the traffic collision is linked to the spatial feature of the capillary instability where capillary and viscous forces acting on the drops evolve along the jet and ultimately amplify small velocity fluctuations. The emergence of such fluctuations is related to the unstable nature of the annular coflow of liquids having contrasting viscoelastic properties. From a practical point of view, flow and actuation conditions can be adjusted to inhibit drop collision and thus drop coalescence. These findings allow then the fabrication of monodisperse submillimeter core-shell objects based on the fragmentation of compound jets made of polymer solutions that find applications for three-dimensional cell culture.

TO THE IPGG TEAMS:

- For any publication having received the support of the IPGG (presence in the IPGG premises, use of the IPGG technological platform, collaboration between IPGG teams, linked to an IPGG doctoral or postdoctoral grant, or use of the common spaces), you must indicate the following sentence : "This work has received the support of "Institut Pierre-Gilles de Gennes" (laboratoire d'excellence, "Investissements d'avenir" program ANR-10-IDEX-0001-02 PSL and ANR-10-LABX-31.) ".

- For any publication of results obtained through the use of equipment purchased by the Equipex IPGG, you must add the following coding: "ANR-10-EQPX-34".

579 publications.