Category
Description
Last updated on 2024-10-04T19:31:35+00:00 by LN Anderson
Activity-Based Probe Fluorescence-Activated Cell Sorting (ABP-FACS) of Soil Microbes (NR-DP0)
Soil samples were incubated with either 0.2% cellobiose for three days or 0.2% xylan for nine days. Microbes were extracted from the soil matrix, incubated overnight in MOPS buffer with either cellobiose or xylose. The following day subsamples from each carbon source incubations were labeled with activity-based probes. For the cellobiose incubated sample, the GH4a probe was used to target glucosidase and cellobiosidase activity, and for the xylan incubated samples, the SYF161 probe was used to target xylosidase and xylobiosidase activity. The GH4a probe was conjugated to a BODIPY (excitation = 502 nm, emission = 511 nm) tag requiring Syto59 (excitation = 622 nm, emission = 645 nm) to be used as the general nucleic acid stain, while SYF161 was conjugated to a Cy5 (excitation = 651 nm, emission = 670 nm) tag and coupled with Syto9 (excitation = 485 nm, emission = 498 nm). The total microbial population, probe unlabeled, and probe labeled were separated based on fluorescence intensity via fluorescence-activated cell sorting (FACS). Samples were collected for direct 16S rRNA sequencing. Additional samples were collected for further incubations in MOPS buffer with either cellobiose or xylose. Aliquots were taken during the post-FACS incubations to track the growth of microbial communities via 16S gene sequencing. Additional negative controls of reagents were collected throughout the process. All samples were sequenced targeting the v4 region of the 16S gene and sequenced using Illumina MiSeq v2 chemistry on a 2x250 flow cell.
Funding Acknowledgments
The research data described here was funded in whole or in part by the Predictive Phenomics Initiative (PPI) at Pacific Northwest National Laboratory (PNNL). This work was conducted under the Laboratory Directed Research and Development Program at PNNL. A portion of this research was performed in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the U.S. Department of Energy (DOE) Office of Science located at PNNL. PNNL is a multiprogram national laboratory operated by Battelle for the DOE under Contract No. DE-AC05-76RL01830.
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Data Licensing
CC BY 4.0 (dataset DOI downloads), CC0 1.0 (PNNL DataHub policy default)
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