Science Core

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Abstract: Science Core, Institut Pasteur The overarching goal of this Core is to supply targeted knock-down mutants, sequencing support and expertise from a centralized facility to the overall Program. The Science Core will be located at the Pasteur Institute in Paris, France. The work of the Science Core will be performed in part at the Pasteur Institute’s, Biology of Spirochetes Unit and in part at the Pasteur Institute’s Biomics Core Facility (C2RT) within the Center for Technological Resources and Research in the Department of Genomics and Genetics. The Science Core will be led by Dr. Marc Monot, who is the director of the Biomics Core Facility. The Biology of Spirochetes Unit is led by Dr. Mathieu Picardeau who is the leading molecular biologist in the leptospiral field and has pioneered new genetic tools for genetically manipulating these fastidious bacteria. The specific objectives of the Science Core are the following: Aim 1 : Engineer targeted mutants. Targeted gene mutants will be created using well-established methods developed by the Biology of Spirochetes Unit including allelic exchange, CRISPR interference (CRISPRi) and Transcription Activator Like Effectors (TALEs). Genes to be targeted include those encoding leucine-rich repeat proteins and dinucleotide cyclases for use by the Coburn and Haake groups, respectively. Aim 2: To perform leptospiral genome sequencing. Leptospiral genome sequencing will be performed on samples isolated from field sites by the Duke project as well as Leptospira strains representative of the diversity of the genus from the collection of Picardeau laboratory and on key transposon and targeted mutants. Sequencing will be performed using short- (Illumina) and long-read (PacBio) technologies. The complete genomes obtained by PacBio sequencing will be used as accurate references for comparative and phylogenetic studies and serve for the analysis of the plasmid content and the methylomes in project 2 (Picardeau). Aim 3: To perform RNA sequencing (RNA-Seq). RNA-stabilized samples to be reverse transcribed and sequenced will include in vitro cultures and hamster samples from the Picardeau project and murine samples (blood and subcutaneous tissue) from the Haake project. RNA-Seq will be also performed on in vitro samples from the Haake project (murine macrophage-Leptospira) and Coburn project (human endothelium-Leptospira). Aim 4: To perform transposon sequencing (Tn-Seq). Tn-Seq will be performed on samples from the Haake and Picardeau projects generated with pools of transposon mutants with known transposon insertion sites. Tn-Seq will be performed by construction of genomic libraries using methods developed by the Haake laboratory including DNA extraction, shearing, and amplification with indexing primers. Picardeau lab will also perform Chromatin immunoprecipitation-sequencing (ChIP-seq) assays for identifying genome-wide DNA binding sites of transcriptional regulators.