A universal pipeline for functional characterization of the human microbiota at a massive scale

项目摘要 我们胃肠道内的微生物群落，统称为肠道微生物区系， 构成了已知的最密集和最多样化的细菌生态系统之一。而亲密的关系 人类和他们的微生物群之间代表着巨大的人类健康工程潜力，我们目前 解开内在复杂性所需的工具有限。我们有能力可预见地利用微生物区系 有益于健康的结果需要对这些细菌的生理学有一个基本的了解，但大多数 人类肠道细菌从未被用分子遗传工具研究过，而且与人类的亲缘关系太远 经过充分研究的模型细菌通过同源性准确地转移基因注释。我们的这一重大差距 对人类肠道细菌基因功能的功能理解必须通过系统的努力来解决， 这将需要多种互补的专业知识。 高通量遗传学是描述基因生物学功能的一种很有吸引力的方法 在人类的微生物区系中。整体扰动在转基因大种群中的应用 细菌允许对几乎所有基因进行平行评估。类似的高吞吐量策略可能会 应用于人类肠道微生物组，但我们的目标是在这方面解决多个主要障碍 项目：(1)非模型细菌的转化仍然具有挑战性，主要是一项试错的努力，(2) 为非模式细菌开发新的遗传系统是耗时的，(3)采用 多项技术和实验室工作流程使跨团队的数据比较变得复杂，(4)在体内 理想情况下，小鼠实验应该在被感兴趣的突变体定居的无菌小鼠身上进行。 为这笔赠款而组建的团队包括处于创新种植方法前沿的领导者， 用于遗传转化的电穿孔，以及用于评估体外和体内基因功能的工具。在目标1中， 我们将迅速开发用于大量人类肠道共生菌株的基因工具，最终目标是 生成全基因组随机条形码转座子突变文库用于测序。我们将利用 这些文库用于在体外测试多种非必需基因的表型重要性(目标2) 和体内(目标3)条件，以全球发现新的基因功能。通过我们在以下领域的综合专业知识 细菌学、微流体、高通量筛选、宿主-微生物相互作用和成像，我们将生产 以前所未有的规模为广大微生物区系研究人员提供遗传工具和健康数据，以及 深入了解人类肠道微生物区系的生理学。

Project Summary The community of microorganisms within our gastrointestinal tract, collectively known as the gut microbiota, constitutes one of the densest and most diverse bacterial ecosystems known. While the close relationship between humans and their microbiota represents vast potential for engineering human health, we are currently limited in tools required to unravel the intrinsic complexity. Our ability to predictably harness the microbiota for beneficial health outcomes requires a fundamental understanding of the physiology of these bacteria, yet most human gut bacteria have never been studied using molecular genetic tools and are too distantly related from well-studied model bacteria to accurately transfer gene annotations by homology. This major gap in our functional understanding of gene functions in human gut bacteria must be addressed with systematic efforts, which will require multiple complementary expertise. High-throughput genetics is an attractive approach for characterizing the biological functions of genes within the human microbiota. Application of perturbations en masse to large populations of genetically modified bacteria permits the parallel assessment of nearly all genes. A similar high-throughput strategy can potentially be applied to the human gut microbiome, but there are multiple major obstacles that we aim to resolve in this project: (1) transformation of non-model bacteria remains challenging and is a largely trial-by-error effort, (2) the development of a new genetic system for a non-model bacterium is time-consuming, (3) the adoption of multiple technologies and laboratory workflows complicates the comparison of data across teams, (4) in vivo mouse experiments should ideally be carried out in ex-germ-free mice colonized by mutants of interest. The team assembled for this grant includes leaders at the forefront of novel cultivation methods, electroporation for genetic transformation, and tools for assessing gene function in vitro and in vivo. In Aim 1, we will rapidly develop genetic tools for a large number of human gut commensal strains, with the ultimate goal of generating genome-wide randomly barcoded transposon mutant libraries for sequencing. We will utilize these libraries to test the phenotypic importance of all non-essential genes across a multitude of in vitro (Aim 2) and in vivo (Aim 3) conditions to globally discover new gene functions. Through our combined expertise in bacteriology, microfluidics, high-throughput screening, host-microbe interactions, and imaging, we will produce genetic tools and fitness data for the vast community of microbiota researchers at unprecedented scale, and deliver deep insight into the physiology of the human gut microbiota.