Use of mCherryOpt Fluorescent Protein in Clostridium difficile

Eric M Ransom; David S Weiss; Craig D Ellermeier

doi:10.1007/978-1-4939-6361-4_5

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Journal article

Use of mCherryOpt Fluorescent Protein in Clostridium difficile

Eric M Ransom, David S Weiss and Craig D Ellermeier

Methods in molecular biology (Clifton, N.J.), Vol.1476, pp.53-67

2016

DOI: 10.1007/978-1-4939-6361-4_5

PMID: 27507333

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Abstract

Here we describe protocols for using the red fluorescent protein mCherryOpt in Clostridium difficile. The protocols can be readily adapted to similar fluorescent proteins (FPs), such as green fluorescent protein (GFP) and cyan fluorescent protein (CFP). There are three critical considerations for using FPs in C. difficile. (1) Choosing the right color: Blue and (especially) red are preferred because C. difficile exhibits considerable yellow-green autofluorescence. (2) Codon optimization: Most FP genes in general circulation have a GC content of ~60 %, so they are not well expressed in low-GC bacteria. (3) Fixing anaerobically grown cells prior to exposure to O2: The FPs under consideration here are non-fluorescent when produced anaerobically because O2 is required to introduce double bonds into the chromophore. Fixation prevents C. difficile cells from becoming degraded during the several hours required for chromophore maturation after cells are exposed to air. Fixation can probably be omitted for studies in which maintaining cellular architecture is not important, such as using mCherryOpt to monitor gene expression.

Conjugation, Genetic

Oxygen - pharmacology

Spectrometry, Fluorescence - methods

Formaldehyde - chemistry

Clostridium difficile - genetics

Green Fluorescent Proteins - genetics

Recombinant Fusion Proteins - metabolism

Codon

Anaerobiosis - genetics

Cloning, Molecular

Escherichia coli - metabolism

Genes, Reporter

Green Fluorescent Proteins - metabolism

Aerobiosis - genetics

Fixatives - chemistry

Plasmids - metabolism

Microscopy, Fluorescence - methods

Base Composition

Escherichia coli - genetics

Plasmids - chemistry

Recombinant Fusion Proteins - genetics

Luminescent Proteins - genetics

Polymers - chemistry

Clostridium difficile - metabolism

Gene Expression Regulation, Bacterial

Clostridium difficile - drug effects

Luminescent Proteins - metabolism

Details

Title: Subtitle: Use of mCherryOpt Fluorescent Protein in Clostridium difficile
Creators: Eric M Ransom - Johne's Testing Center, School of Veterinary Medicine, Madison, WI, USA
David S Weiss - Department of Microbiology, University of Iowa, 51 Newton Rd, 3-372 Bowen Science Building, Iowa City, IA, 52242, USA
Craig D Ellermeier - Department of Microbiology, University of Iowa, 51 Newton Rd, 3-372 Bowen Science Building, Iowa City, IA, 52242, USA. craig-ellermeier@uiowa.edu
Resource Type: Journal article
Publication Details: Methods in molecular biology (Clifton, N.J.), Vol.1476, pp.53-67
Publisher: United States
DOI: 10.1007/978-1-4939-6361-4_5
PMID: 27507333
eISBN: 1493963619; 9781493963614
ISSN: 1064-3745
eISSN: 1940-6029
Grant note: R01 AI087834 / NIAID NIH HHS
Language: English
Date published: 2016
Academic Unit: Microbiology and Immunology
Record Identifier: 9984001204202771

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