Science Core

科学核心

基本信息

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
项目类别：
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

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

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项目摘要

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，研究所的巴斯德该核心的总体目标是提供有针对性的敲门突变体，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上进行 Spirochetes单元，部分是巴斯德研究所的生物核心核心设施（C2RT）基因组学和遗传学系的技术资源和研究。科学核心将由生物学核心设施主任马克·莫诺特（Marc Monot）博士领导。螺旋体单元的生物学是由Mathieu Picardeau博士领导，他是钩端螺旋体领域的领先分子生物学家新的遗传工具，用于操纵这些挑剔的细菌。科学核心的特定对象如下：目标1：工程师靶向突变体。靶向基因突变体将使用良好的方法创建由Spirochetes单元的生物学开发转录激活因子（Tales）。靶向靶向的基因包括编码丰富亮氨酸的基因分别重复蛋白质和二核苷酸循环，分别由玉米粉和Haake组使用。目标2：执行钩端螺旋体基因组测序。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的钩端螺旋体菌株 Picardeau实验室和关键转座子和靶向突变体的属的多样性。测序将使用短（Illumina）和长阅读（PACBIO）技术进行。完整通过PACBIO测序获得的基因组将用作比较和系统发育研究并用于分析项目2中的质粒含量和甲基组（Picardeau）。目标3：执行RNA测序（RNA-Seq）。 RNA稳定的样品要进行反转录，并测序将包括Picardeau项目的体外培养物和仓鼠样品和鼠类样品（血液和皮下组织）来自Haake项目。 RNA-seq还将在体外样品上进行来自Haake Project（鼠巨噬细胞）和Coburn Project（人类内皮 - 肠道皮）。目标4：执行转座测序（TN-SEQ）。 TN-Seq将在Haake的样本上进行， Picardeau项目由带有已知转座插入位点的转座子突变体池产生。 tn-seq 将通过使用Haake实验室开发的方法来构建基因组库来执行包括DNA提取，剪切和索引引物的扩增。 Picardeau Lab也将表演用于鉴定全基因组DNA结合位点的染色质免疫沉淀 - 测定（CHIP-SEQ）转录调节器。