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

基本信息

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
项目类别：
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

项目摘要

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.

该副本是利用众多研究子项目之一由NIH/NCRR资助的中心赠款提供的资源。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是对于中心，这不一定是调查员的机构。 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在含有自动荧光的物种的复杂混合物中。 LANL流式细胞仪和细胞分选的最新进展（提高的分选能力，相位仪器和声学焦点）可以克服当前使用流量排序的当前局限性用于收集和评估土壤和微生物垫等复杂环境的微生物成分。当前的研究应用在生物治疗，气候变化和基因组学中可以受益于从复杂混合物中特异性物种或功能组的特定物种或功能组的能力。例子包括从气溶胶中分离未培养的弗朗西斯菌细胞，土壤或MAT群落的光合作用和非光合合成成员的分配，以及在宏观分析进行复杂微生物群落之前的活性/不活跃成分的分馏。我们建议与项目1上的流资源合作，应用声学焦点和分类技术来回答环境微生物生态学中的基本问题，我们为这项技术提供了三种应用，并按照越来越复杂和困难的顺序提出。我们将使用这三个项目来帮助推动声学焦点技术的开发及其与现有流动技术的集成。土壤微生物群落由多种原核生物（细菌）和真核（真菌，藻类，微肢体）组成。对于土壤微生物学中的许多应用，需要将一组与另一组分离。由于两种细胞类型代表的各种形状和尺寸，目前的技术都无法成功执行此操作。由于它基于大小和形状的实时分离，并且具有非常广泛的动态范围，因此声学焦点技术具有将复杂的微生物混合物分离为尺寸类别的潜力，其次是核定/无核域，可以帮助我们丰富社区的特定组成部分。该能力的直接应用包括在元基因组分析之前降低复杂性，以及对已知在其中一个域表示已知的特定目标基因或官能团的敏感性。第二个潜在应用是从空气样品中对复杂的微生物混合物进行排序。我们目前对美国城市中数千个空气过滤器的评估（通过与Biowch和EPA的计划）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，我们试图从多种形成孢子的物种中分离出尚不可培养的特定靶标细菌病原体。声学焦点技术将使我们能够将目标细胞与“污染”孢子分开。在先前尝试使用流式细胞术和排序以丰富复杂环境混合物中特定的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，从而使荧光染色的细胞与未染色的细胞进行分离，而使用传统流动细胞仪和分类困难。声学焦点通过形状和大小对细胞群体进行分类的能力，以及在天然自动荧光光谱以外的波长中可检测到的灯笼染料的掺入，使其成为富集的潜在强大工具，可用于特定染色的微生物组件。在许多情况下，这种直接富集是下游基因组分析的最有希望的方法。