Confining Single Cells to Enhance and Target Cultivation of Human Microbiome

描述（由申请人提供）：了解人类微生物组对于维持人类健康和预防疾病至关重要，但由于大多数微生物无法使用传统方法培养，因此尚不清楚特定微生物如何影响健康和疾病。需要既能提高培养微生物的成功率，又能将培养目标对准高生物医学价值的微生物的技术。本项目将通过开发“单细胞约束技术”，利用微流体约束克服传统培养和靶向方法的局限性。将单个细胞随机限制在小体积（皮升到纳升）的液滴中，将分离微生物物种，并有可能通过从单个细胞开始高密度生长来培养新的微生物。这种单细胞约束技术产生的液滴可以分裂，在克隆姐妹群体上并行进行多项检测，从而可以在一个姐妹群体中进行杀死试验，以识别微生物，并使用另一个姐妹群体进行生长。在这项技术中，为了将培养目标对准具有生物医学意义的微生物，将通过两种互补的方法来鉴定新物种：基于基因的检测和基于功能的检测。基于基因的分析，根据现有的宏基因组数据，将识别所需的功能基因和16S序列，基于功能的分析将识别所需的功能，即使它们与已知的基因序列不相关。鉴定出的微生物物种将成为扩大微菌落规模的目标，使它们可用于测序和进一步研究。我们将利用人类结肠中的硫还原菌作为测试系统，开发和验证单细胞约束技术。硫还原菌在生物医学上具有重要意义，与溃疡性结肠炎和腹腔内感染有关，但对其了解甚少。我们将首先使用肠道衍生微生物的模型财团，包含代表性的硫还原细菌嗜硫杆菌wadsworthia，以开发和优化该技术。接下来，我们将开发基于基因和功能的检测方法，并通过鉴定模型混合物中的硫酸盐还原菌来测试它们。最后，我们将使用这些培养方法和检测方法从人类结肠中培养和选择新的硫还原细菌。这项技术将普遍适用于鉴定和培养人类肠道微生物组中的所有类别的微生物。该项目将通过开发和验证技术来提高我们对人类肠道微生物群中基因和功能之间关系的理解，从而影响生物医学科学和公共卫生，因此微生物对健康和疾病的贡献。

DESCRIPTION (provided by applicant): Understanding the human microbiome is critical in maintaining human health and preventing disease, but it has been unclear how specific microbes affect health and disease because the majority of microbes cannot be cultivated using traditional methods. Technologies are needed that can both increase the success rate for cultivating microbes and target cultivation efforts towards microbes of high biomedical interest. This project will use microfluidic confinement to overcome the limitations of traditional cultivation and targeting methods by developing "single cell confinement technology". Stochastic confinement of single cells in droplets of small volumes (picoliters to nanoliters) will isolate microbial species and, potentially, enable cultivation of new microbes by initiating high-density growth from a single cell. The droplets created by this single cell confinement technology can be split to perform multiple assays in parallel on clonal sister populations, enabling killing assays to be performed to identify microbes in one sister population, and using of the other sister population for growth. In this technology, to target cultivation efforts towards microbes of biomedical interest, new species will be identified via two complementary approaches: gene-based assays and function-based assays. Gene-based assays, informed by existing metagenomic data, will identify desired functional genes and 16S sequences, and function-based assays will identify desired functions even if they are not associated with a known gene sequence. The identified microbial species will then be targeted for scale-up of microcolonies to make them available for sequencing and further study. We will develop and validate the single cell confinement technology by using sulfur-reducing bacteria from the human colon as the test system. Sulfur-reducing bacteria are of high biomedical importance, associated with Ulcerative Colitis and intra-abdominal infections, but are still poorly understood. We will first use a model consortium of gut-derived microorganisms, containing a representative sulfur-reducing bacterium Bilophila wadsworthia, to develop and optimize the technology. Next, we will develop gene-based and function- based assays and test them by identifying sulfate reducing bacteria in model mixtures. Finally, we will use these cultivation approaches and assays to cultivate and select new sulfur-reducing bacteria from the human colon. This technology will be generally applicable to identify and cultivate all classes of microbes in the human gut microbiome. This project will impact biomedical science and public health by developing and validating technologies for increasing our understanding of the relationship between genes and functions in the human gut microbiome, and therefore microbial contributions to both health and disease. PUBLIC HEALTH RELEVANCE: Statement Microbes are critical to the function of the gastrointestinal tract. Understanding their role in human health and disease requires cultivation, but the majority of the species in the human gut microbiome are difficult to cultivate. This application will develop confined-based technology to enhance cultivation of microbes from human colon and target the cultivation efforts by using complementary assays to identify microbes with genes and functions of high biomedical interest.