SORTING CELLS IN COMPLEX ENVIRONMENTS FOR FUNCTIONAL AND GENOMIC ANALYSIS

在复杂环境中对细胞进行分类以进行功能和基因组分析

• 批准号: 8169408
• 负责人: Cheryl R Kuske
• 金额: $ 1.67万
• 依托单位: LOS ALAMOS NAT SECTY-LOS ALAMOS NAT LAB
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169408
• 关键词: Acoustics; Aerosols; Algae; Bacteria; Cell Separation; Cells; Cities; Collection; Communities; Complex; Complex Mixtures; Computer Retrieval of Information on Scientific Projects Database; Detection; Development; Dyes; Ecology; Environment; Evaluation; Flow Cytometry; Fractionation; Francisella; Funding; Gene Targeting; Genomics; Grant; Individual; Institution; Lanthanoid Series Elements; Metagenomics; Methods; Microbe; Phase; Population; Reproduction spores; Research; Research Personnel; Resources; Shapes; Soil; Soil Microbiology; Sorting - Cell Movement; Source; Staining method; Stains; Techniques; Technology; Time; United States National Institutes of Health; air filter; air sampling; base; biothreat; cell type; climate change; functional genomics; functional group; fungus; improved; instrument; member; microbial; microbial community; microorganism; pathogen; programs; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The ability to sort and individually interrogate specific fractions of microorganisms in the environment is hampered by the high diversity of microbes (bacteria, fungi, algae, and potentially millions of species), by our inability to culture the vast majority of microorganisms that can be observed microscopically, and by the lack of hybridization methods that are sensitive enough to detect cells that are not metabolically active or that are present in complex mixtures containing species that are autofluorescent. Recent advances in flow cytometry and cell sorting at LANL (improved sorting capability, phase instruments and acoustic focusing) could overcome some of the current limitations in use of flow sorting for collection and evaluation of microbial components of complex environments such as soils and microbial mats. Current research applications in biothreat detection, climate change, and genomics could benefit from the ability to specifically sort individual species or functional groups of bacteria out of complex mixtures. Examples include isolation of uncultured Francisella cells from aerosols, partitioning of photosynthetic and non-photosynthetic members of soil or mat communities, and fractionation of active/inactive components of complex microbial communities prior to metagenomic analysis. We propose to collaborate with the Flow Resource on Project 1, to apply the acoustic focusing and sorting technology to answer fundamental questions in environmental microbial ecology, and we present three applications for this technology, presented in order of increasing sophistication and difficulty. We will use these three projects to help drive the development of the acoustic focusing technology and its integration with existing flow techniques. Soil microbial communities are comprised of a wide variety of prokaryotic (bacteria) and eukaryotic (fungi, algae, microarthropods) assemblages. For many applications in soil microbiology it is desirable to isolate one group from the other. No current technology can successfully do this due to the wide variety of shapes and sizes represented by both cell types. Because it separates in real time, based on both size and shape, and has a very broad dynamic range, the acoustic focusing technology has potential to separate complex microbial mixtures into size classes, followed by nucleated/anucleated domains, that can help us enrich for particular components of the community. Immediate applications for this capability include reducing complexity prior to metagenome analysis, and increasing sensitivity for particular target genes or functional groups known to be represented in one of the domains. A second potential application, is to sort complex microbial mixtures from air samples. Our current evaluations of thousands of air filters in U.S. cities (through programs with BioWatch and the EPA), have documented a diverse array of bacterial species present in respirable aerosols. In some cases, we are trying to isolate particular target bacterial pathogens that are not-yet-culturable, from an abundance of spore-forming species. The acoustic focusing technology would allow us to separate the target cells from the 'contaminating' spores. In previous attempts to use flow cytometry and sorting to enrich for specific unculturable bacteria from complex environmental mixtures, we have been hampered by the tendency of different bacterial species to form aggregates, making separation of fluorescently stained vs. unstained cells difficult using tradition flow cytometry and sorting. The ability of the acoustic focusing to sort cell populations by shape as well as size, and the incorporation of lanthanide dyes that are detectable at wavelengths outside the natural autofluorescence spectrum, make it a potentially powerful tool to enrich for specifically stained microbial components. In many cases, such direct enrichment is the most promising approach for downstream genomic analysis.

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 环境中微生物的高度多样性阻碍了对微生物的特定组分进行分类和单独询问的能力（细菌，真菌，藻类，以及潜在的数百万物种），由于我们无法培养绝大多数可以在显微镜下观察到的微生物，以及缺乏足够灵敏的杂交方法来检测不具有代谢活性的细胞或存在于含有物种的复杂混合物中的细胞是自发荧光的。LANL在流式细胞术和细胞分选方面的最新进展（改进的分选能力，相位仪器和声学聚焦）可以克服目前使用流式分选收集和评估复杂环境（如土壤和微生物垫）中微生物组分的一些限制。目前在生物威胁检测、气候变化和基因组学方面的研究应用可以受益于从复杂的混合物中特异性地分选出单个物种或细菌功能群的能力。实例包括从气溶胶中分离未培养的弗朗西斯菌细胞，划分土壤或垫群落的光合和非光合成员，以及在宏基因组分析之前分离复杂微生物群落的活性/非活性组分。 我们建议与流动资源项目1合作，应用声学聚焦和分选技术来回答环境微生物生态学中的基本问题，我们提出了这项技术的三个应用，按照复杂性和难度的增加顺序提出。 我们将利用这三个项目来帮助推动声聚焦技术的发展及其与现有流动技术的整合。 土壤微生物群落由多种原核生物（细菌）和真核生物（真菌、藻类、微型节肢动物）组成。对于土壤微生物学中的许多应用，期望将一个组与另一个组分离。由于这两种细胞类型所代表的形状和大小的多样性，目前没有技术可以成功地做到这一点。因为它基于大小和形状在真实的时间内分离，并且具有非常宽的动态范围，所以声聚焦技术具有将复杂的微生物混合物分离成大小类别的潜力，然后是有核/无核域，这可以帮助我们富集群落的特定组分。这种能力的直接应用包括在宏基因组分析之前降低复杂性，以及增加已知在其中一个结构域中代表的特定靶基因或官能团的灵敏度。 第二个潜在的应用是从空气样品中分选复杂的微生物混合物。我们目前对美国城市中数千个空气过滤器的评估（通过BioWatch和EPA的项目）记录了可吸入气溶胶中存在的各种细菌物种。在某些情况下，我们试图从大量形成孢子的物种中分离出尚未培养的特定目标细菌病原体。声聚焦技术将使我们能够将目标细胞与“污染”孢子分离。 在先前使用流式细胞术和分选从复杂环境混合物中富集特定不可培养细菌的尝试中，我们受到不同细菌物种形成聚集体的趋势的阻碍，使得使用传统流式细胞术和分选难以分离荧光染色的细胞与未染色的细胞。声聚焦通过形状和大小对细胞群进行分类的能力，以及在天然自发荧光光谱之外的波长处可检测到的镧系元素染料的掺入，使其成为富集特异性染色的微生物组分的潜在有力工具。在许多情况下，这种直接富集是下游基因组分析最有前途的方法。