Science Core

科学核心

• 批准号: 10643289
• 负责人: Marc Monot
• 金额: $ 6.05万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-16 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10643289
• 关键词: Alleles; Bacteria; Binding Proteins; Binding Sites; Bioinformatics; Biological Assay; Biology; Blood; CRISPR interference; Cadherins; ChIP-seq; Collection; Complement; Core Facility; DNA; DNA Binding; Development; Dinucleoside Phosphates; Endothelium; Engineering; Genes; Genetic; Genetic Transcription; Genome; Genomic Library; Genomics; Goals; Hamsters; Human; In Vitro; Individual; International; Knock-out; Laboratories; Leptospira; Leptospirosis; Libraries; Macrophage; Methods; Methyltransferase; Molecular; Mus; Order Spirochaetales; Oxidative Stress; Paris, France; Phylogenetic Analysis; Plasmids; Proteins; RNA; Research; Resources; Reverse Transcription; Sampling; Science; Services; Site; Subcutaneous Tissue; Technology; Transcription Coactivator; Work; comparative; genetic manipulation; genome database; genome sequencing; genome-wide analysis; indexing; innovation; knock-down; leucine-rich repeat protein; methylome; mutant; pathogen; programs; success; tool; transcriptome sequencing; transcriptomics; transposon sequencing

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.

摘要：科学核心，巴斯德研究所 该核心的总体目标是提供有针对性的击倒突变体、测序支持和专业知识 从集中设施到整体计划。科学核心将位于巴斯德研究所 法国巴黎。科学核心的工作将部分在巴斯德研究所的生物学中心进行 螺旋体单位和巴斯德研究所生物组学核心设施（C2RT）的一部分 基因组学和遗传学系的技术资源和研究。科学核心将 由生物组学核心设施主任 Marc Monot 博士领导。螺旋体生物学单位是 由Mathieu Picardeau博士领导，他是钩端螺旋体领域的领先分子生物学家，并开创了 用于对这些挑剔细菌进行基因操作的新遗传工具。 科学核心的具体目标如下： 目标 1：设计目标突变体。将使用成熟的方法创建目标基因突变体 由螺旋体生物学单位开发，包括等位基因交换、CRISPR 干扰 (CRISPRi) 和 转录激活因子样效应器 (TALE)。目标基因包括编码富含亮氨酸的基因 重复蛋白质和二核苷酸环化酶分别供 Coburn 和 Haake 小组使用。 目标 2：进行钩端螺旋体基因组测序。钩端螺旋体基因组测序将在 杜克大学项目从现场分离出的样本以及代表钩端螺旋体菌株 来自皮卡多实验室收藏的该属的多样性以及关键转座子和目标突变体。 将使用短读长 (Illumina) 和长读长 (PacBio) 技术进行测序。完整的 通过PacBio测序获得的基因组将作为准确的参考进行比较和 系统发育研究并用于项目2中质粒内容和甲基化组的分析 （皮卡多）。 目标 3：进行 RNA 测序 (RNA-Seq)。 RNA 稳定的样品进行反转录和 测序将包括皮卡多项目的体外培养物和仓鼠样本以及小鼠样本 （血液和皮下组织）来自哈克项目。 RNA-Seq 也将在体外样本上进行 来自 Haake 项目（鼠巨噬细胞-钩端螺旋体）和 Coburn 项目（人内皮细胞-钩端螺旋体）。 目标 4：进行转座子测序 (Tn-Seq)。 Tn-Seq 将对 Haake 和 Picardeau 项目利用具有已知转座子插入位点的转座子突变体池生成。肌钙蛋白测序 将通过使用 Haake 实验室开发的方法构建基因组文库来进行 包括 DNA 提取、剪切和使用索引引物的扩增。皮卡多实验室也将表演 用于识别全基因组 DNA 结合位点的染色质免疫沉淀测序 (ChIP-seq) 分析 转录调节因子。