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Hydrocarbon and Lipid Microbiology Protocols pp 163–180Cite as

Generating Enriched Metagenomes from Active Microorganisms with DNA Stable Isotope Probing

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Abstract

Cultivation-dependent methods have long been used to characterise the physiology of microorganisms after they have been isolated from the environment. In contrast, cultivation-independent methods help better identify the microorganisms that regulate specific biogeochemical processes in situ, regardless of their ability to grow in the laboratory. In this chapter, we describe how the cultivation-independent technique of DNA stable isotope probing (DNA-SIP) can be combined with targeted functional gene probing and metagenomic analyses to characterise the metabolic potential of active microbes from environmental samples, including those that assimilate labelled hydrocarbons. DNA-SIP enables the separation of DNA from microbes that have assimilated a given 13C-labelled substrate into their biomass (i.e., heavy DNA) from those that have not (i.e., light DNA). The heavy DNA can be used subsequently for PCR amplicon sequencing and metagenome sequencing, which can potentially lead to retrieving genomes of uncultivated and active microbial representatives.

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Acknowledgements

This work was possible thanks to the financial support from the Gordon and Betty Moore Foundation Marine Microbiology Initiative Grant GBMF3303 to J. Colin Murrell and Yin Chen, and the Earth and Life Systems Alliance, Norwich Research Park, Norwich, UK. Josh Neufeld acknowledges a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC).

Author information

Authors and Affiliations

  1. School of Environmental Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK

    Carolina Grob, Martin Taubert, Alexandra M. Howat & J. Colin Murrell

  2. School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK

    Oliver J. Burns

  3. School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK

    Yin Chen

  4. Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, N2L 3G1, Canada

    Josh D. Neufeld

Authors
  1. Carolina Grob
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  2. Martin Taubert
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  3. Alexandra M. Howat
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  4. Oliver J. Burns
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  5. Yin Chen
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  6. Josh D. Neufeld
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  7. J. Colin Murrell
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Corresponding author

Correspondence to J. Colin Murrell .

Editor information

Editors and Affiliations

  1. Dept. Biological Sciences, University of Essex Dept. Biological Sciences, Colchester, Essex, United Kingdom

    Terry J. McGenity

  2. Braunschweig, Germany

    Kenneth N. Timmis

  3. Department of Biology, University of the Balearic Islands and Mediterranean Institute for Advanced Studies (IMEDEA, UIB-CSIC), Palma de Mallorca, Spain

    Balbina Nogales

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© 2015 Springer-Verlag Berlin Heidelberg

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Grob, C. et al. (2015). Generating Enriched Metagenomes from Active Microorganisms with DNA Stable Isotope Probing. In: McGenity, T., Timmis, K., Nogales , B. (eds) Hydrocarbon and Lipid Microbiology Protocols. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8623_2015_81

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  • DOI: https://doi.org/10.1007/8623_2015_81

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  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-662-50449-9

  • Online ISBN: 978-3-662-50450-5

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