Abstract
Chip-SIP is a stable isotope probing (SIP) method for linking microbial identity and function in mixed communities and is capable of analyzing multiple isotopes (13C, 15N, and 18O) simultaneously. This method uses a high-density microarray to separate taxon-specific 16S (or 18S) rRNA genes and a high sensitivity magnetic sector secondary ion mass spectrometer (SIMS) to determine the relative isotope incorporation of the rRNA at each probe location. Using a maskless array synthesizer (MAS), we synthesize multiple unique sequences to target hundreds of taxa at the ribosomal operational taxonomic unit (OTU) level on an array surface, and then analyze it with a NanoSIMS 50, using its high-spatial resolution imaging capability to generate isotope ratios for individual probes. The Chip-SIP method has been used in diverse systems, including surface marine and estuarine water, rhizosphere, and peat soils, to quantify taxon-specific relative incorporation of different substrates in complex microbial communities. Depending on the hypothesis and experimental design, Chip-SIP allows the user to compare the same community incorporating different substrates, different communities incorporating the same substrate(s), or quantify how a community responds to treatment effects, such as temperature or nutrient concentrations.
Key words
- Stable isotope probing
- NanoSIMS
- 16S rRNA
- Microbial ecology
- Microarrays
- 13C
- 15N
- 18O
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Acknowledgments
The work leading to the results presented in the chapter was performed under the auspices of the US Department of Energy at the Lawrence Livermore National Laboratory under Contract DEAC52-07NA27344. Funding provided by the Genomic Sciences Program from DOE-OBER through the Biofuels Science Focus Area Grant SCW1039 and the microbial carbon cycle program grant # SCW1590. LLNL’s Laboratory Directed Research and Development Program (07-ERD-053) and the Austrian Science Fund (FWF P23797 P27275 and P30596) is acknowledged.
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Mayali, X. et al. (2019). Chip-SIP: Stable Isotope Probing Analyzed with rRNA-Targeted Microarrays and NanoSIMS. In: Dumont, M., Hernández García, M. (eds) Stable Isotope Probing. Methods in Molecular Biology, vol 2046. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9721-3_6
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DOI: https://doi.org/10.1007/978-1-4939-9721-3_6
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