PAPM EAGER: Microwell array platform for high-throughput screening and discovery of microbial interactions

该项目旨在开发一种新的工具，用于发现与根相关的细菌群落中发生的相互作用。数千种不同的细菌长期生活在植物根部，细菌的相互作用有助于形成这些群落，是决定植物健康的关键因素。传统的方法一次只能测试少量的相互作用，导致许多细菌群落特征不明显。通过同时测试数千种不同的细菌相互作用，我们的方法将大大加快发现的步伐。发现这些相互作用将有助于操纵细菌群落，以改善食品生产和环境净化工作。我们的方法将是高度适应性的，允许检查任何微生物群落和在任何微生物实验室使用。该项目涉及一个由工程师和微生物学家组成的跨学科团队，他们将应用微生物生态学和遗传学概念在微加工方面的最新进展，从而为博士后研究员和研究生提供跨学科培训。此外，该项目将支持弗林特山探索中心的一个互动公共推广项目，说明土地利用管理实践对弗林特山生态区微生物群多样性的影响，以及如何利用微纳米技术提高我们对微生物的理解。微生物之间的相互作用影响微生物群落的动态、组成和对宿主的影响。本项目旨在开发一种高通量筛选方法，用于鉴定影响局灶细菌种类的细菌种类。筛选平台将使用微孔阵列在焦点物种和微生物组内不同细菌物种之间创建数千种独特的配对。使用聚合物膜将细胞对困在各自的孔中，然后用荧光显微镜监测对焦点物种的影响。将提取显示增强或抑制焦点物种功能的井，并对拮抗或促进物种进行测序以进行鉴定。我们将在康扎长期生态研究（LTER）站点的不同土地利用管理制度下取样两组向日葵（Helianthus annuus）微生物组，验证该技术平台。第一次筛选将确定拮抗或促进通用植物病原体农杆菌生长的细菌。为了确定微生物相互作用的具体机制，第二个筛选将确定微生物组成员，这些微生物组成员可以积极或消极地影响famefaciens群体感应系统的诱导。建立的平台将是低成本的，操作简单，并适用于任何微生物组的发现。

This project aims to develop a new tool for the discovery of interactions occurring within root-associated bacterial communities. Thousands of different bacteria persistently live on plant roots where bacterial interactions help shape these communities and are a critical factor in determining plant health. Traditional approaches test only a few interactions at a time, leaving many bacterial communities poorly characterized. By simultaneously testing thousands of different bacterial interactions, our approach will greatly accelerate the pace of discovery. Uncovering these interactions will aid efforts to manipulate bacterial communities to improve food production and environmental decontamination efforts. Our method will be highly adaptable, allowing for examination of any microbial community and for use in any microbiology laboratory. The project involves an interdisciplinary team of engineers and microbiologists that will apply recent advances in microfabrication with concepts from microbial ecology and genetics, thereby providing interdisciplinary training for a post-doctoral researcher and a graduate student. In addition, the project will support an interactive public outreach program at the Flint Hills Discovery Center illustrating the impact of land use management practices on microbiome diversity in the Flint Hills ecoregion, as well as how micro- and nanotechnologies can be used to improve our understanding of microbes.Microbe-microbe interactions influence microbial community dynamics, composition, and impact on the host. This project aims to develop a high-throughput screening approach for identifying bacterial species that impact a focal bacterial species. The screening platform will use a microwell array to create thousands of unique pairings between the focal species and different bacterial species within a microbiome. Cell pairs will be trapped within their respective wells using a polymer membrane and then monitored with a fluorescent microscope for effects on the focal species. Wells showing enhancement or suppression of focal species function will be extracted and the antagonizing or promoting species will be sequenced for identification. We will validate this technology platform with two screens of Helianthus annuus microbiomes sampled from sites under different land use management regimes at the Konza Long-Term Ecological Research (LTER) site. The first screen will identify bacteria that antagonize or promote the growth of the generalist plant pathogen Agrobacterium tumefaciens. To target specific mechanisms of microbial interaction, the second screen will identify microbiome members that either positively or negatively influence induction of A. tumefaciens' quorum sensing system. The established platform will be low-cost, simple to operate, and applicable for discovery in any microbiome.