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.

翻译后摘要：科学核心，巴斯德研究所 该核心的首要目标是提供靶向敲除突变体，测序支持和专业知识 从一个集中的设施到整个项目。科学核心将位于巴斯德研究所， 法国，巴黎。科学核心的部分工作将在巴斯德研究所的生物学中心进行。 螺旋体单位和部分在巴斯德研究所的生物学核心设施（C2 RT）内的中心， 基因组学和遗传学系的技术资源和研究。科学核心将 由Marc Monot博士领导，他是生物学核心设施的主任。螺旋体生物学单位是 由马蒂厄·皮卡多博士领导，他是钩端螺旋体领域的领先分子生物学家， 新的遗传工具来遗传操纵这些挑剔的细菌。 科学核心的具体目标如下： 目标1：设计目标突变体。靶向基因突变体将使用完善的方法创建 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子（TALE）。靶向基因包括那些编码富含亮氨酸的 重复蛋白和二核苷酸环化酶，分别供Coburn和Haake组使用。 目的2：进行钩端螺旋体全基因组测序。钩端螺旋体基因组测序将在 由杜克项目从现场分离的样品以及代表性的钩端螺旋体菌株 Picardeau实验室收集的属的多样性以及关键转座子和靶向突变体。 测序将使用短读（Illumina）和长读（PacBio）技术进行。完整的 通过PacBio测序获得的基因组将用作比较和 系统发育研究，并为项目2中的质粒含量和甲基化组分析服务 （Picardeau）. 目的3：进行RNA测序（RNA-Seq）。待逆转录的RNA稳定化样品， 测序将包括体外培养物和来自Picardeau项目的仓鼠样本以及小鼠样本 （血液和皮下组织）从哈克项目。还将对体外样本进行RNA-Seq 来自Haake项目（鼠巨噬细胞-钩端螺旋体）和Coburn项目（人内皮-钩端螺旋体）。 目的4：进行转座子测序（Tn-Seq）。将对来自Haake的样本进行Tn-Seq， Picardeau项目用具有已知转座子插入位点的转座子突变体库产生。Tn-Seq 将通过使用Haake实验室开发的方法构建基因组文库进行 包括DNA提取、剪切和用索引引物扩增。皮卡多实验室还将执行 用于鉴定全基因组DNA结合位点的染色质免疫沉淀测序（ChIP-seq）测定 转录调节因子的作用。