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Detection of Protein-Synthesizing Microorganisms in the Environment via Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT)

Hatzenpichler, Roland and Orphan, Victoria J. (2015) Detection of Protein-Synthesizing Microorganisms in the Environment via Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT). In: Hydrocarbon and Lipid Microbiology Protocols: Single-Cell and Single-Molecule Methods. Springer , Berlin, pp. 145-157. ISBN 978-3-662-49129-4.

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Bioorthogonal noncanonical amino acid tagging (BONCAT) is a recently developed method for studying microbial in situ activity. This technique is based on the in vivo incorporation of artificial amino acids that carry modifiable chemical tags into newly synthesized proteins. BONCAT has been demonstrated to be effective in labeling the proteomes of a wide range of taxonomically and physiologically distinct Archaea and bacteria without resulting in preferential synthesis or degradation of proteins. After chemical fixation of cells, surrogate-containing proteins can be detected by whole-cell fluorescence staining using azide-alkyne click chemistry. When used in conjunction with rRNA-targeted fluorescence in situ hybridization (FISH), BONCAT allows the simultaneous taxonomic identification of a microbial cell and its translational activity. Rather than studying the bulk proteome, BONCAT is able to specifically target proteins that have been expressed in reaction to an experimental condition. BONCAT-FISH thus provides researchers with a selective, sensitive, fast, and inexpensive fluorescence microscopy technique for studying microbial in situ activity on an individual cell level. This protocol provides a detailed description of how to design and perform BONCAT experiments using two different bioorthogonal amino acids, l-azidohomoalanine (AHA) and l-homopropargylglycine (HPG), which are both surrogates of l-methionine. It illustrates how incorporation of these noncanonical amino acids into new proteins can be detected via copper-catalyzed or strain-promoted azide-alkyne click chemistry and outlines how the visualization of translational activity can be combined with the taxonomic identification of cells via FISH. Last, the protocol discusses potential problems that might be encountered during BONCAT studies and how they can be overcome.

Item Type:Book Section
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URLURL TypeDescription
Hatzenpichler, Roland0000-0002-5489-3444
Orphan, Victoria J.0000-0002-5374-6178
Additional Information:© 2015 Springer-Verlag Berlin Heidelberg. First Online: 04 April 2015.
Group:Division of Geological and Planetary Sciences
Subject Keywords:AHA; Anabolic activity; Bioorthogonal chemistry; Click chemistry; Ecophysiology; FISH; HPG; Protein synthesis; Single cell; Translation
Record Number:CaltechAUTHORS:20170714-110726557
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:79111
Deposited By: Tony Diaz
Deposited On:14 Jul 2017 18:32
Last Modified:23 Jan 2023 19:33

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