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：巴斯德研究所科学核心 这个核心的首要目标是提供有针对性的敲除突变体、测序支持和专业知识 从一个集中设施到整个计划。科学核心将设在#年的巴斯德研究所 法国巴黎。科学核心的部分工作将在巴斯德研究所的 螺旋体单位，部分在巴斯德研究所的生物组学核心设施(C2RT)内 基因组学与遗传学系的技术资源与研究。科学核心将 由Marc Monot博士领导，他是生物组学核心设施的主任。螺旋体单位的生物学是 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 用新的基因工具对这些挑剔的细菌进行基因操作。 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 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(TALS)。靶向基因包括编码富含亮氨酸的基因 分别由Coburn和Haake小组使用的重复蛋白和二核苷酸环酶。 目的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 杜克项目从现场分离的样本以及具有代表性的钩端螺旋体菌株 来自Picardeau实验室收集的属的多样性以及关键转座子和目标突变体的多样性。 将使用短读(Illumina)和长读(PacBio)技术进行测序。完整的 通过PacBio测序获得的基因组将被用作比较和 进行系统发育研究，并用于分析项目2中的质粒物和甲基组 (皮卡多)。 目的3：进行RNA测序(RNA-Seq)。RNA稳定的样本将被逆转录和 测序将包括来自Picardeau项目的体外培养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(小鼠巨噬细胞-钩端螺旋体)和Coburn项目(人类内皮-钩端螺旋体)。 目的4：进行转座子测序(TN-Seq)。TN-Seq将对来自Haake和 Picardeau项目是用已知转座子插入位点的转座子突变体池产生的。TN-Seq 将通过使用哈克实验室开发的方法构建基因组文库来执行 包括DNA的提取、剪切和用索引引物进行扩增。皮卡多实验室也将表演 染色质免疫沉淀测序(ChIP-SEQ)方法鉴定全基因组DNA结合位点 转录调控因子。