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URL: https://dx.doi.org/DOI:10.17504/protocols.io.utkewkw

Authors: Jackie Goordial

Group: Orcutt Deep Biosphere Lab

Summary: BONCAT is a technique that allows for the visualization and isolation of metabolically active cells from complex environmental enrichments. This is achieved by incubating a sample with synthetic methionine analogs AHA (l-azidohomoalanine) or HPG (l-homopropargylglycine). Instead of the sulfhydryl side group of methionine , these analogs contain an azide or alkyne functional group, respectively, which can be chemically tagged with a fluorophore using a process termed “click-chemistry”. Throughout the incubation period actively growing cells incorporate these synthetic amino acids into newly synthesized proteins. After harvesting cells from a live enrichment, the cells are tagged with the fluorophore. This allows for microscopic visualization along with FACS-based isolation of the metabolically active cells in a community. Downstream analysis can include single-cell sequencing or metagenomics techniques to determine taxonomy and functions of isolated organisms. Notes: Working with low-biomass samples has an inherent high-risk of contamination. Be mindful of sterility at all times. Perform all work in the biological safety cabinet and make sure to sterilize the hood with UV prior beginning work Some of the BONCAT reagents are light-sensitive, be careful to pay attention to if a particular step in the protocol needs to be performed in the darkThe click-chemictry reaction is very sensitive to oxygen, so take care not to vortex/ introdroduce air unnecessarily Try to plan when you harvest incubations so that you are certain you will be able to perform the click-chemistry process the next morning

Affiliations: orcutt lab protocols

Version: 1

Publication Date: 2018

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