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

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.