Experimental workshops

WORKSHOP: Eukaryotic Molecular Biology

Vinciane Regnier, India Leclercq, Jean-François Ouimette, Guillaume Velasco

Identification of genomic and transcriptomic targets of a transcription factor in a lymphocyte context

• Mapping of transcription factor binding sites on the human genome

DNA sequencing data analysis (ChIP-seq)

-Introduction to the Galaxy platform (https://usegalaxy.org/) and sequencing data analysis tools in the context of a ChIP-seq experiment (Immunoprecipitation of chromatin followed by “high throughput” sequencing).

– Development of a “pipeline” for the analysis and identification of genomic targets of a transcription factor from public ChIP-seq data.

• Transcription factor and gene expression: experimental approaches

– Realization of a ChIP experiment using lymphoblastoid lines. Analysis by ChIP-qPCR of the presence of the transcription factor of interest at the level of sequences identified by the bioinformatics study (Galaxy)Realization of a ChIP experiment using lymphoblastoid lines. Analysis by ChIP-qPCR of the presence of the transcription factor of interest at the level of sequences identified by the bioinformatics study (Galaxy)

– RT-qPCR analysis of gene expression near the transcription factor binding site in a “wild” or “mutant” context for the transcription factor under study

Workshop Virulence factors and host immune response

Anne Couedel-Courteille – Ignacio Garcia-Verdugo

To explore bacterial/immune cell interactions in terms of virulence factors and host response, through a transversal approach rich in techniques used in the fields of biochemistry, cellular and molecular biology, enzymology, bacteriology and immunology.

To study the impact of bacterial virulence factors important for pathogenicity in the recognition of the bacterium by the host immune system and its response

• Biochemistry techniques (western blot),
• Cell biology techniques (cell culture, fluorescence microscopy),
• Enzymology techniques (enzymatic assay, zymogram),
• Bacteriology techniques (growth curves, plate spreading),
• Immunology techniques (phagocytosis, ELISA) to study bacterial virulence factors important for pathogenicity and recognition of the bacteria by the host immune system.

WORKSHOP: IntegraProt

Catherine Etchebest, Julien Dairou, Wilfried Grange, Karine Moncoq, Stéphanie Petrella, Thérèse de Caldas

Integrative study of a eukaryotic enzymatic system.
Integrated structure-function approach to characterize glycogen phosphorylase b, an allosterically regulated enzyme.

• Exploration of the family of this protein and its structural properties using the Chimera tool.
• Prediction of functional sites based on multiple alignments. Study of the impact of mutations.
• Preparation of a crude cell extract and purification of glycogen phosphorylase b by affinity chromatography and SDS-PAGE gel analysis.
• Demonstration of the allosteric properties of the enzyme (using an effector) and the important role of certain residues (by chemical labelling) in this regulation (initial speed measurements and data processing using specific software and/or the R language).
• Study of ligand binding by spectrofluorometry and demonstration of energy transfer.
• Visualization and structure-function analysis using Pymol software.