DNA Stable Isotope Probing Mehod to Examine Carbon Assimilation in Stream Biofilms
Location
Logan Country Club
Start Date
3-28-2017 2:15 PM
End Date
3-28-2017 2:20 PM
Description
DNA stable isotope probing (DNA-SIP) identifies microbial populations that assimilate a substrate of interest by tracking the movement of an isotopic label from a substrate into DNA. Although the technique has not been widely used in streams, DNA-SIP can be used to identify populations in stream biofilms that use different sources of carbon. We performed a DNA-SIP experiment to identify populations that assimilate mannitol and algal carbon. Stream biofilms grown on glass discs were incubated in a growth chamber in river water amended with 13C-mannitol or 13C-bicarbonate for 3 and 10 days. Biofilm DNA was extracted and separated by density using ultracentrifugation. To identify populations that assimilated our two carbon sources, we collected the heavier, isotopically-labeled DNA and sequenced the 16S rRNA gene. Sequences were analyzed using MOTHUR, operational taxonomic units identified using Megablast, and phylogenetic identities aligned against the Greengenes database. In addition to carbon assimilation, future studies can use DNA-SIP to identify populations contributing to nitrogen cycling and other stream biogeochemical processes, enhancing understanding of the link between microbial identity and ecosystem function.
DNA Stable Isotope Probing Mehod to Examine Carbon Assimilation in Stream Biofilms
Logan Country Club
DNA stable isotope probing (DNA-SIP) identifies microbial populations that assimilate a substrate of interest by tracking the movement of an isotopic label from a substrate into DNA. Although the technique has not been widely used in streams, DNA-SIP can be used to identify populations in stream biofilms that use different sources of carbon. We performed a DNA-SIP experiment to identify populations that assimilate mannitol and algal carbon. Stream biofilms grown on glass discs were incubated in a growth chamber in river water amended with 13C-mannitol or 13C-bicarbonate for 3 and 10 days. Biofilm DNA was extracted and separated by density using ultracentrifugation. To identify populations that assimilated our two carbon sources, we collected the heavier, isotopically-labeled DNA and sequenced the 16S rRNA gene. Sequences were analyzed using MOTHUR, operational taxonomic units identified using Megablast, and phylogenetic identities aligned against the Greengenes database. In addition to carbon assimilation, future studies can use DNA-SIP to identify populations contributing to nitrogen cycling and other stream biogeochemical processes, enhancing understanding of the link between microbial identity and ecosystem function.