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:
    8361772
  • 负责人:
  • 金额:
    $ 1.12万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2011
  • 资助国家:
    美国
  • 起止时间:
    2011-04-01 至 2013-03-31
  • 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. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.
这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的, 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量, 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 由于微生物的高度多样性(细菌、真菌、藻类,可能还有数以百万计的物种),我们无法培养绝大多数可以通过显微镜观察到的微生物,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自体荧光物种的复杂混合物中,阻碍了对环境中特定微生物部分进行分类和单独询问的能力。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、相仪器和声聚焦)可以克服目前使用流动分选收集和评估土壤和微生物垫等复杂环境中微生物成分的一些限制。目前在生物修复检测、气候变化和基因组学方面的研究应用可能受益于从复杂混合物中具体分离单个细菌物种或功能组的能力。例如,从气溶胶中分离未培养的Francisella细胞,划分土壤或垫子群落的光合作用和非光合作用成员,以及在进行元基因组分析之前对复杂微生物群落的活性/非活性成分进行分离。 我们建议与Flow Resources在项目1上合作,应用声聚焦和分类技术来回答环境微生物生态学中的基本问题,并按复杂性和难度递增的顺序介绍了该技术的三个应用。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、微节肢动物)组成。在土壤微生物学的许多应用中,将一个组与另一个组分离是可取的。由于两种细胞类型所代表的形状和大小多种多样,目前还没有一种技术能够成功地做到这一点。由于声聚焦技术根据大小和形状实时分离,并具有非常宽的动态范围,因此有可能将复杂的微生物混合物分离成大小类别,然后是有核/无核域,这可以帮助我们丰富群落的特定组成部分。这种能力的直接应用包括在元基因组分析之前降低复杂性,以及提高对已知在其中一个结构域中代表的特定靶基因或功能基团的敏感性。 第二个潜在的应用是从空气样本中对复杂的微生物混合物进行分类。我们目前对美国城市数千个空气过滤器的评估(通过与BioWatch和EPA的项目)记录了可呼吸气雾剂中存在的各种细菌种类。在某些情况下,我们正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。声波聚焦技术将使我们能够将目标细胞与受污染的孢子分开。 在以前使用流式细胞仪和分选来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倾向的阻碍,使得使用传统的流式细胞术和分选分离荧光染色的细胞和未染色的细胞变得困难。声聚焦按形状和大小对细胞群进行分类的能力,以及在自然自发荧光光谱以外的波长上可检测到的稀土染料的加入,使其成为一种潜在的强大工具,用于浓缩特定染色的微生物成分。在许多情况下,这种直接浓缩是最有希望的下游基因组分析方法。

项目成果

期刊论文数量(0)
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Cheryl R Kuske其他文献

Cheryl R Kuske的其他文献

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{{ truncateString('Cheryl R Kuske', 18)}}的其他基金

PEPTIDE INTERACTION MECHANISM WITH LAMELLAR LIPID BILAYERS: VARIANTS OF C3A PEPT
肽与层状脂质双层的相互作用机制:C3A PEPT 的变体
  • 批准号:
    8362174
  • 财政年份:
    2011
  • 资助金额:
    $ 1.12万
  • 项目类别:
PEPTIDE INTERACTION MECHANISM WITH LAMELLAR LIPID BILAYERS: VARIANTS OF C3A PEPT
肽与层状脂质双层的相互作用机制:C3A PEPT 的变体
  • 批准号:
    8170125
  • 财政年份:
    2010
  • 资助金额:
    $ 1.12万
  • 项目类别:
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
  • 财政年份:
    2010
  • 资助金额:
    $ 1.12万
  • 项目类别:
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:
    7956791
  • 财政年份:
    2009
  • 资助金额:
    $ 1.12万
  • 项目类别:
PEPTIDE INTERACTION MECHANISM WITH LAMELLAR LIPID BILAYERS: VARIANTS OF C3A PEPT
肽与层状脂质双层的相互作用机制:C3A PEPT 的变体
  • 批准号:
    7954455
  • 财政年份:
    2009
  • 资助金额:
    $ 1.12万
  • 项目类别:
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:
    7724270
  • 财政年份:
    2008
  • 资助金额:
    $ 1.12万
  • 项目类别:
PEPTIDE INTERACTION MECHANISM WITH LAMELLAR LIPID BILAYERS: VARIANTS OF C3A PEPT
肽与层状脂质双层的相互作用机制:C3A PEPT 的变体
  • 批准号:
    7722151
  • 财政年份:
    2008
  • 资助金额:
    $ 1.12万
  • 项目类别:

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