Engineering ‘Plug-and-Play’ Cell-Free Biosensors

Cell-free protein synthesis represents a promising avenue for developing next-generation diagnostics. Researchers at the Center for Structural Biology in Montpellier and the Michalis Institute in Jouy-en-Josas recently expanded the potential of these 'cell-free biosensors' by combining transcription factor-based detection with metabolic engineering. The synthetic biosensors are capable of detecting benzoic acid in commercial beverages and hippuric acid and cocaine in human urine. These results have been published in the journal Nature Communications along with a 'Behind the Scenes' article for the Nature Bioengineering Community.

cell free biosensors

Transcription and chromosome observation in single cells with Hi-M

Direct and simultaneous observation of transcription and chromosome architecture in single cells with Hi-M

Andrés M. Cardozo Gizzi, Sergio M. Espinola, Julian Gurgo, Christophe Houbron, Jean-Bernard Fiche, Diego I. Cattoni, Marcelo Nollmann

Simultaneous observation of 3D chromatin organization and transcription at the single cell level and with high spatial resolution may hold the key to unveil the mechanisms regulating embryonic development, cell differentiation and even disease. We have recently developed Hi-M, a technology that allows for the sequential labelling, 3D imaging and localization of multiple genomic DNA loci together with RNA expression in single cells within whole, intact Drosophila embryos. Importantly, Hi-M enables simultaneous detection of RNA expression and chromosome organization without requiring sample unmounting and primary probe re-hybridization. Here, we provide a step-by-step protocol describing the design of probes, the preparation of samples, the stable immobilization of embryos into microfluidics chambers, and the complete procedure for image acquisition. The combined RNA/DNA fluorescence in situ hybridization procedure takes 4-5 days including embryo collection. In addition, we describe image analysis software to segment nuclei, detect genomic spots, correct for drift and produce Hi-M matrices. A typical Hi-M experiment takes 1-2 days to complete all rounds of labelling and imaging and 4 additional days for image analysis. This technology can be easily expanded to investigate cell differentiation in cultured cells, or organization of chromatin within complex tissues.

Nat Protoc 2020 Mar;15(3):840-876. doi: 10.1038/s41596-019-0269-9


Study on cis-regulatory chromatin loops

Cis-regulatory chromatin loops arise before TADs and gene activation, and are independent of cell fate during development

Sergio Martin Espinola, Markus Götz, Jean-Bernard Fiche, Maelle Bellec, Christophe Houbron, Andrés M. Cardozo Gizzi, Mounia Lagha, Marcelo Nollmann

During development, naïve cells gradually acquire distinct cell fates, through sophisticated mechanisms of precise spatio-temporal gene regulation. Acquisition of cell fate is thought to rely on the specific interaction of remote *cis*-regulatory modules (e.g. enhancers, silencers) (CRM) and target promoters. However, the precise interplay between chromatin structure and gene expression is still unclear, particularly in single cells within multicellular developing organisms. Here we employ Hi-M, a single-cell spatial genomics approach, to systematically detect CRM-promoter looping interactions within topological associating domains (TADs) during *Drosophila* development. By comparing *cis*-regulatory loops in alternate cell types, we show that physical proximity does not necessarily instruct transcriptional states. Moreover, multi-way analyses revealed the existence of local interactions between multiple remote CRMs to form hubs. We found that loops and CRM hubs are established early during development, prior to the emergence of TADs. Moreover, CRM hubs are formed via the action of the pioneer transcription factor Zelda and precede transcriptional activation. Our approach offers a new perspective on the role of CRM-promoter interactions in defining transcriptional activation and repression states, as well as distinct cell types.


Insights into the "cocktail effect" of endocrine disruptors

Chemicals which taken in isolation are safe for humans may become harmful when mixed . The team of William Bourguet in Structural Biochemistry Center (Inserm / CNRS / University of Montpellier), together with teams from the Cancer Research Institute (IRCM ) and the Functional Genomics Institute (IGF ) in Montpellier elucidated in vitro a molecular mechanism that may contribute to this phenomenon known as the "cocktail effect".

New publication: "Synergistic activation of human pregnane X receptor by binary cocktails of pharmaceutical and environmental compounds"
Authors: Delfosse V, Dendele B, Huet T, Grimaldi M, Boulahtouf A, Gerbal-Chaloin S, Beucher B, Roecklin D, Muller C, Rahmani R, Cavaillès V, Daujat-Chavanieu M, Vivat V, Pascussi JM, Balaguer P, Bourguet W.
Journal: Nat Communication 2015 Sep 3;6:8089.


New Structure of a Protein Modulating Bacterial Gene Silencing

Antibiotic resistance and the appearance of new virulent bacterial strains constitute a major threat to human health. The problem is aggravated by the transfer of resistance and virulence genes between bacteria (horizontal gene transfer). In this context, a detailed knowledge of the mechanisms allowing bacteria to tolerate the acquisition of foreign DNA is lacking and it may open the way to new sustainable strategies to fight infectious diseases infectious diseases.

Here we describe a structural model for the complex between Hha and H-NS proteins which selective represses genes in Enterobacteria acquired by horizontal transfer. We found a charge zipper formed by interdigitation of residues from three proteins stabilizes the complex. Charge zippers provide selectivity to electrostatic protein complexes and understanding selective gene silencing may help fighting antibiotic resistance

New publication: "A Three-protein Charge Zipper Stabilizes a Complex Modulating Bacterial Gene Silencing"
Authors: Tiago N. Cordeiro, Jesús García, Pau Bernadó, Oscar Millet et Miquel Pons
Journal: Biol Chem. 2015 Aug 28;290(35):21200-12
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