In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry.
In situ visualization of newly synthesized proteins in environmental microbes using amino acid tagging and click chemistry.
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DOI：
10.1111/1462-2920.12436
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发表时间：
2014-08
影响因子：
5.1
通讯作者：
Orphan VJ
中科院分区：
文献类型：
--
作者：
Hatzenpichler R;Scheller S;Tavormina PL;Babin BM;Tirrell DA;Orphan VJ
Here we describe the application of a new click chemistry method for fluorescent tracking of protein synthesis in individual microorganisms within environmental samples. This technique, termed bioorthogonal non-canonical amino acid tagging (BONCAT), is based on the in vivo incorporation of the non-canonical amino acid L-azidohomoalanine (AHA), a surrogate for l-methionine, followed by fluorescent labelling of AHA-containing cellular proteins by azide-alkyne click chemistry. BONCAT was evaluated with a range of phylogenetically and physiologically diverse archaeal and bacterial pure cultures and enrichments, and used to visualize translationally active cells within complex environmental samples including an oral biofilm, freshwater and anoxic sediment. We also developed combined assays that couple BONCAT with ribosomal RNA (rRNA)-targeted fluorescence in situ hybridization (FISH), enabling a direct link between taxonomic identity and translational activity. Using a methanotrophic enrichment culture incubated under different conditions, we demonstrate the potential of BONCAT-FISH to study microbial physiology in situ. A direct comparison of anabolic activity using BONCAT and stable isotope labelling by nano-scale secondary ion mass spectrometry (15NH3 assimilation) for individual cells within a sediment-sourced enrichment culture showed concordance between AHA-positive cells and 15N enrichment. BONCAT-FISH offers a fast, inexpensive and straightforward fluorescence microscopy method for studying the in situ activity of environmental microbes on a single-cell level.
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DOI：
10.1007/978-1-61779-151-2_5
发表时间：
2011-01-01
期刊：
BIOCONJUGATION PROTOCOLS: STRATEGIES AND METHODS, SECOND EDITION
影响因子：
--
作者：
Banerjee, Partha S.;Carrico, Isaac S.
通讯作者：
Carrico, Isaac S.
影响因子：
4.4
作者：
Behrens, Sebastian;Loesekann, Tina;Spormann, Alfred M.
通讯作者：
Spormann, Alfred M.
影响因子：
3.2
作者：
Beatty, Kimberly E.;Fisk, John D.;Smart, Brian P.;Lu, Ying Ying;Szychowski, Janek;Hangauer, Matthew J.;Baskin, Jeremy M.;Bertozzi, Carolyn R.;Tirrell, David A.
通讯作者：
Tirrell, David A.
影响因子：
30.3
作者：
Chakrabarti, Sveta;Liehl, Peter;Lemaitre, Bruno
通讯作者：
Lemaitre, Bruno
影响因子：
56.9
作者：
Dekas, Anne E.;Poretsky, Rachel S.;Orphan, Victoria J.
通讯作者：
Orphan, Victoria J.