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Confining Single Cells to Enhance and Target Cultivation of Human Microbiome

限制单细胞以增强和定向人类微生物组的培养

基本信息

批准号：
8523446
负责人：
RUSTEM F ISMAGILOV
金额：
$ 18.35万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-08-01 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8523446
关键词：
Abdominal Infection Address Affect Bilophila wadsworthia Biological Assay Biopsy Specimen Cells Colon Communities Data Daughter Development Disease Gases Gastrointestinal tract structure Genes Genetic Genome Growth Health Human Human Microbiome Individual Inorganic Sulfates Intra-abdominal Life Liquid substance Metagenomics Methodology Methods Microbe Microfluidics Modeling Organism Pathway interactions Persons Population Population Growth Production Public Health Role Science Screening procedure Sister Solid Sulfur-Reducing Bacteria System Technology Testing Ulcerative Colitis Unspecified or Sulfate Ion Sulfates Work acrosome stabilizing factor base cell killing data sharing density gene function improved interest killings microbial microbial community microbiome microorganism nanolitre new technology prevent public health relevance scale up success sulfate reducing bacteria technology development

项目摘要

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.

了解人体微生物组对于维护人类健康和预防疾病至关重要，但目前尚不清楚特定微生物如何影响健康和疾病，因为大多数微生物无法使用传统方法培养。需要既能提高培养微生物的成功率又能将培养工作目标对准具有高度生物医学意义的微生物的技术。该项目将利用微流体限制技术，通过开发“单细胞限制技术”克服传统培养和靶向方法的局限性。将单细胞随机限制在小体积（皮升至纳升）的液滴中将分离微生物物种，并且可能通过从单细胞开始高密度生长来培养新的微生物。通过这种单细胞限制技术产生的液滴可以被分裂以在克隆姐妹群体上并行进行多个测定，使得能够进行杀伤测定以鉴定一个姐妹群体中的微生物，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：基于基因的测定和基于功能的测定。基于基因的测定，由现有的宏基因组数据告知，将识别所需的功能基因和16S序列，基于功能的测定将识别所需的功能，即使它们与已知的基因序列无关。然后将确定的微生物物种作为小菌落规模扩大的目标，使其可用于测序和进一步研究。我们将开发和验证的单细胞禁闭技术，通过使用硫还原菌从人类结肠作为测试系统。硫还原菌具有很高的生物医学重要性，与溃疡性结肠炎和腹腔内感染有关，但仍然知之甚少。我们将首先使用一个模型财团的肠道衍生的微生物，含有一个代表性的硫还原细菌Bilophila wadsaproia，开发和优化的技术。接下来，我们将开发基于基因和基于功能的测定，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测定来培养和选择来自人类结肠的新的硫还原细菌。这项技术将普遍适用于识别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影响生物医学科学和公共卫生，以增加我们对人类肠道微生物组中基因和功能之间关系的理解，从而影响微生物对健康和疾病的贡献。公共卫生相关性：声明微生物对胃肠道功能至关重要。了解它们在人类健康和疾病中的作用需要培养，但人类肠道微生物组中的大多数物种很难培养。该应用将开发基于限制的技术，以增强人类结肠微生物的培养，并通过使用互补测定来鉴定具有高度生物医学兴趣的基因和功能的微生物，从而靶向培养工作。