Quantifying Horizontal Gene Transfer in the Human Gut Microbiome

量化人类肠道微生物组中的水平基因转移

• 批准号: 10246308
• 负责人: Benjamin Emery Rubin
• 金额: $ 6.56万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10246308
• 关键词: Antibiotic Resistance; Attention; Base Sequence; Biological Assay; Biology; Categories; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complex; DNA; DNA delivery; Data; Data Set; Development; Drug resistance; Ecology; Effectiveness; Engineering; Event; Experimental Models; Foundations; Gene Transfer; Genes; Genetic; Genetic Materials; Genome; Genomics; Health; Horizontal Gene Transfer; Human; Human Microbiome; In Situ; Life; Link; Maps; Measures; Memory; Methods; Microbe; Mobile Genetic Elements; Modeling; Modification; Molecular; Molecular Biology; Molecular Cloning; Movement; Mutation; Pathway interactions; Penetrance; Plasmids; Process; RNA; Resources; Role; Route; System; Techniques; Testing; Training; Travel; Virulence; Virulence Factors; Visit; Work; base; bioinformatics pipeline; cohort; comparative genomics; cost; design; genetic manipulation; gut microbiome; gut microbiota; human microbiota; improved; in vitro Model; insight; integration site; interest; member; microbial; microbial community; microbiome; microorganism; pathogen; permissiveness; resistance factors; success; tool; vector

Project Summary Horizontal gene transfer (HGT) in the human microbiome has considerable impact on human health through its role in disseminating virulence factors and antibiotic resistance. Due to the complexity of studying HGT within microbiomes, however, it has received limited experimental attention. This proposal characterizes dynamics of HGT in the gut microbiome through the development and application of new experimental tools. We will begin by optimizing a technique developed in our preliminary work called ET-Seq, which serves as a sequence-based tool to measure permissiveness of microbial community members to HGT agents of interest. The approach will be applied to test the ability of human microbiota members to take up DNA from various forms of HGT. Agents for conjugation, transduction, and transformation will be assayed in parallel by ET-Seq for their efficiency against the microbial constituents of a realistic in vitro model of the human gut microbiome. Using comparative genomics, the community landscape of permissiveness and restrictiveness to the different means of HGT will be linked to the genes responsible. We will also supplement ET-Seq's static snapshot of the state of HGT by creating molecular recordings of HGT. To accomplish this, we will add CRISPR-Cas based molecular recorders to HGT agents so that they record their trajectory through the gut microbiota by picking up host DNA as they travel. These routes of HGT will later be recovered by sequencing of the HGT agent's recording cassettes. Together these approaches will provide a dynamic and comprehensive view of HGT in the human gut microbiome. This work has potential for far reaching human health impacts. HGT is extremely prevalent in the gut and serves to spread drug resistance and virulence genes. Understanding how HGT occurs should provide data necessary to mitigate this subset of undesirable events. Furthermore, this work will provide a foundation for targeted editing within the gut microbiome. It will elucidate the effectiveness of various gene transfer agents on gut microbiota members and provide the tools necessary to track genetic changes implemented in this, or any other community. The computational and molecular ecology skillsets of Jill Banfield's lab, the molecular biology and CRISPR-Cas expertise of Jennifer Doudna's lab, and the gut microbiome models of Melanie Ott's lab will provide broad resources for the success of this project as well as extensive training potential.

项目摘要 人类微生物组中的水平基因转移（HGT）通过其对人类健康的影响， 传播毒力因子和抗生素耐药性的作用。由于研究HGT的复杂性， 然而，微生物组，它受到了有限的实验关注。这一建议的特点是动态的 HGT在肠道微生物组中通过开发和应用新的实验工具。 我们将开始通过优化在我们的初步工作中开发的称为ET-Seq的技术， 基于序列的工具来测量微生物群落成员对感兴趣的HGT试剂的容许性。 该方法将用于测试人类微生物群成员从各种微生物中摄取DNA的能力。 HGT的形式。用于缀合、转导和转化的试剂将通过ET-Seq平行测定。 因为它们对人类肠道微生物组的真实体外模型的微生物组分的有效性。 利用比较基因组学，社区景观的宽容和限制，以不同的 HGT的方式将与相关基因联系起来。我们还将补充ET-Seq的静态快照， 通过创建HGT的分子记录来观察HGT的状态。为了实现这一目标，我们将添加基于CRISPR-Cas的 分子记录器，以便他们记录他们的轨迹通过肠道微生物群， 携带DNA的病毒这些HGT途径随后将通过对HGT试剂的DNA序列进行测序来恢复。 录音带。这些方法将共同提供一个动态和全面的HGT视图， 人类肠道微生物组。 这项工作有可能对人类健康产生深远的影响。HGT在肠道中非常普遍， 传播抗药性和毒力基因。了解HGT如何发生应提供数据 这是缓解这类不良事件所必需的。此外，这项工作将为以下方面奠定基础： 在肠道微生物组内进行有针对性的编辑。它将阐明各种基因转移剂对 肠道微生物群成员，并提供必要的工具，以跟踪在此实施的遗传变化，或任何 其他社区。吉尔·班菲尔德实验室的计算和分子生态学技能， Jennifer Doudna实验室的CRISPR-Cas专业知识，以及Melanie Ott实验室的肠道微生物组模型， 为该项目的成功提供广泛的资源以及广泛的培训潜力。