A fluorescent reporter for single cell analysis of gene expression in clostridium difficile
A fluorescent reporter for single cell analysis of gene expression in clostridium difficile
Genetically identical cells growing under homogeneous growth conditions often display cell-cell variation in gene expression. This variation stems from noise in gene expression and can be adaptive allowing for division of labor and bet-hedging strategies. In particular, for bacterial pathogens, the expression of phenotypes related to virulence can show cell-cell variation. Therefore, understanding virulence-related gene expression requires knowledge of gene expression patterns at the single cell level. We describe protocols for the use of fluorescence reporters for single cell analysis of gene expression in the human enteric pathogen Clostridium difficile, a strict anaerobe. The reporters are based on modified versions of the human DNA repair enzyme O ( 6)-alkylguanine-DNA alkyltransferase, called SNAP-tag and CLIP-tag. SNAP becomes covalently labeled upon reaction with O ( 6)-benzylguanine conjugated to a fluorophore, whereas CLIP is labeled by O ( 6)-benzylcytosine conjugates. SNAP and CLIP labeling is orthogonal allowing for dual labeling in the same cells. SNAP and CLIP cassettes optimized for C. difficile can be used for quantitative studies of gene expression at the single cell level. Both the SNAP and CLIP reporters can also be used for studies of protein subcellular localization in C. difficile.
Aldehydes/metabolism, Anaerobiosis/genetics, Base Sequence, Benzyl Compounds/chemistry, Clostridioides difficile/genetics, Cytosine/chemistry, Fluorescent Dyes/chemistry, Gene Expression Regulation, Bacterial, Genes, Reporter, Guanine/analogs & derivatives, Humans, Indoles/metabolism, Microscopy, Fluorescence, O(6)-Methylguanine-DNA Methyltransferase/genetics, Peptides/chemistry, Plasmids/chemistry, Pyridinium Compounds/metabolism, Quaternary Ammonium Compounds/metabolism, Single-Cell Analysis/methods, Staining and Labeling/methods
69-90
Cassona, Carolina Piçarra
500e22e3-39ec-45bf-972c-5be593d9b021
Pereira, Fátima
a9396948-26f9-4f13-8f83-a22fec1dd0e0
Serrano, Mónica
131f2fee-4325-486b-aeee-6a9be8c00ed6
Henriques, Adriano O
eb4668e6-bfb0-4df3-bf8e-93dbd1cc5b2d
10 August 2016
Cassona, Carolina Piçarra
500e22e3-39ec-45bf-972c-5be593d9b021
Pereira, Fátima
a9396948-26f9-4f13-8f83-a22fec1dd0e0
Serrano, Mónica
131f2fee-4325-486b-aeee-6a9be8c00ed6
Henriques, Adriano O
eb4668e6-bfb0-4df3-bf8e-93dbd1cc5b2d
Cassona, Carolina Piçarra, Pereira, Fátima, Serrano, Mónica and Henriques, Adriano O
(2016)
A fluorescent reporter for single cell analysis of gene expression in clostridium difficile.
Methods in molecular biology (Clifton, N.J.), 1476, .
(doi:10.1007/978-1-4939-6361-4_6).
Abstract
Genetically identical cells growing under homogeneous growth conditions often display cell-cell variation in gene expression. This variation stems from noise in gene expression and can be adaptive allowing for division of labor and bet-hedging strategies. In particular, for bacterial pathogens, the expression of phenotypes related to virulence can show cell-cell variation. Therefore, understanding virulence-related gene expression requires knowledge of gene expression patterns at the single cell level. We describe protocols for the use of fluorescence reporters for single cell analysis of gene expression in the human enteric pathogen Clostridium difficile, a strict anaerobe. The reporters are based on modified versions of the human DNA repair enzyme O ( 6)-alkylguanine-DNA alkyltransferase, called SNAP-tag and CLIP-tag. SNAP becomes covalently labeled upon reaction with O ( 6)-benzylguanine conjugated to a fluorophore, whereas CLIP is labeled by O ( 6)-benzylcytosine conjugates. SNAP and CLIP labeling is orthogonal allowing for dual labeling in the same cells. SNAP and CLIP cassettes optimized for C. difficile can be used for quantitative studies of gene expression at the single cell level. Both the SNAP and CLIP reporters can also be used for studies of protein subcellular localization in C. difficile.
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Published date: 10 August 2016
Keywords:
Aldehydes/metabolism, Anaerobiosis/genetics, Base Sequence, Benzyl Compounds/chemistry, Clostridioides difficile/genetics, Cytosine/chemistry, Fluorescent Dyes/chemistry, Gene Expression Regulation, Bacterial, Genes, Reporter, Guanine/analogs & derivatives, Humans, Indoles/metabolism, Microscopy, Fluorescence, O(6)-Methylguanine-DNA Methyltransferase/genetics, Peptides/chemistry, Plasmids/chemistry, Pyridinium Compounds/metabolism, Quaternary Ammonium Compounds/metabolism, Single-Cell Analysis/methods, Staining and Labeling/methods
Identifiers
Local EPrints ID: 470699
URI: http://eprints.soton.ac.uk/id/eprint/470699
ISSN: 1064-3745
PURE UUID: cff26da7-12cd-4e57-aa73-b40758aac421
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Date deposited: 18 Oct 2022 16:41
Last modified: 17 Mar 2024 04:14
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Contributors
Author:
Carolina Piçarra Cassona
Author:
Fátima Pereira
Author:
Mónica Serrano
Author:
Adriano O Henriques
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