Second Generation Stable Isotope Probing Technologies: Solid Phase DNA array measures coupled to Stable Isotope functional Tracers.

第二代稳定同位素探测技术：固相 DNA 阵列测量与稳定同位素功能示踪剂相结合。

• 批准号: NE/I001093/1
• 负责人: Andrew Whiteley
• 金额: $ 13.47万
• 依托单位: NERC CEH (Up to 30.11.2019)
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2010
• 资助国家: 英国
• 起止时间: 2010 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=NE%2FI001093%2F1
• 关键词: Second; Generation; Stable; Isotope; Probing

Identifying organisms and understanding how they work is a central pre-requisite for biology. For large organisms (e.g. animals and plants), direct observation has been a cornerstone of understanding biology and ecology for centuries. More recently, these observational studies have been significantly enhanced through the use of molecular biology to study DNA and understand how genes contribute to the biology of an organism. However, when we look microscopic organisms, e.g. bacteria, observational studies are limited due to their small size and lack of morphological diversity, and thus, critical information has been primarily derived from studies which exclusively target the organism's DNA. This is especially true for microbes such as bacteria, where the natural environment harbours an enormous diversity of species and functions, but of which we have little understanding of how they actually work. Assessment of how microbial populations work is critical since they provide many of the basic ecosystem services (element cycling, pollution clean-up, plant growth promotion) which help to run planet Earth. Several issues exist in using DNA based approaches to study bacteria. First, bacteria in the environment are incredibly diverse, upwards of 10,000,000 species have been estimated globally and 10,000 species probably reside in a gram of soil, hence we need methods that are quick and easy to implement. Identifying genes are good handles on the types of organisms and their genes which are present in a sample, but they give little true indication of the actual function a bacterial species is performing. A species or gene may simply be present in a sample but the gene is not being used for a variety of reasons. Therefore, we need systems which can rapidly identify species and genes in a sample, but also link this identification to actual functions we can measure within the sample. One way for doing this is to use labelled tracers which act as a 'bait' and which have a unique marker that we can detect within the species/genes associated with the process. For example, if we wished to look at carbon cycling, we can use stable isotope labelled carbon ('heavy' 13C carbon as opposed to 12C 'normal' carbon) which will specifically label those organisms which are involve in processing carbon. The extra neutron in 'heavy' 13C provides a unique signature which we can measure and can be therefore used to detect the functionally active organisms, since they will label with the 13C based marker. Our aim is to combine rapid gene identification and detection of the unique signatures of isotopes by combining DNA array technology, which can detect upwards of 500,000 genes in a single analysis over a few hours, with stable isotope measurements of the genes themselves within the DNA array. This will allow us to sub-divide those species/genes that are labelled and those that are not, and hence identify the 'functionally active' species in the environment. We will rapidly detect labelled species using new developments in spectroscopy which provide unique 'fingerprints' for stable isotope labelled molecules versus non-labelled molecules and combine both DNA array and spectroscopy analysis in a single measurement assay.

识别生物体并了解它们是如何工作的是生物学的核心先决条件。对于大型生物（如动物和植物），几个世纪以来，直接观察一直是理解生物学和生态学的基石。最近，这些观察性研究通过使用分子生物学来研究DNA并了解基因如何对生物体的生物学做出贡献而得到了显着增强。然而，当我们观察微生物（如细菌）时，由于其体积小且缺乏形态多样性，观察性研究受到限制，因此，关键信息主要来自专门针对生物体DNA的研究。对于细菌这样的微生物来说尤其如此，自然环境孕育着物种和功能的巨大多样性，但我们对它们的实际工作原理知之甚少。评估微生物种群如何工作是至关重要的，因为它们提供了许多基本的生态系统服务（元素循环、污染清理、促进植物生长），有助于地球的运行。使用基于DNA的方法研究细菌存在几个问题。首先，环境中的细菌种类繁多，全球估计有超过1000万种细菌，一克土壤中可能有1万种细菌，因此我们需要快速简便的方法。识别基因可以很好地处理样品中存在的生物体及其基因的类型，但它们对细菌物种的实际功能几乎没有真正的指示。一个物种或基因可能只是存在于样本中，但由于各种原因，该基因没有被使用。因此，我们需要能够快速识别样品中的物种和基因的系统，但也将这种识别与我们可以在样品中测量的实际功能联系起来。一种方法是使用标记示踪剂作为“诱饵”，它具有独特的标记，我们可以在与该过程相关的物种/基因中检测到。例如，如果我们希望研究碳循环，我们可以使用稳定同位素标记的碳（“重”13C碳，而不是12C“正常”碳），这将专门标记那些参与碳加工的生物。“重”13C中的额外中子提供了一个独特的特征，我们可以测量它，因此可以用来检测功能活跃的生物，因为它们会用基于13C的标记来标记。我们的目标是通过结合DNA阵列技术，将快速基因鉴定和同位素独特特征的检测结合起来，DNA阵列技术可以在几个小时内检测到50万个以上的基因，并在DNA阵列中对基因本身进行稳定的同位素测量。这将使我们能够细分那些被标记的物种/基因和那些没有标记的物种/基因，从而识别环境中“功能活跃”的物种。我们将利用光谱学的新发展快速检测标记物种，该技术为稳定同位素标记分子与非标记分子提供独特的“指纹”，并将DNA阵列和光谱分析结合在一次测量分析中。

项目成果

Chemical fixation methods for Raman spectroscopy-based analysis of bacteria.

• DOI: 10.1016/j.mimet.2014.12.008
• 发表时间: 2015-02
• 期刊: Journal of microbiological methods
• 影响因子: 2.2
• 作者: D. Read;A. Whiteley