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Appl Environ Microbiol. 2015 Oct;81(20):7244-52. doi: 10.1128/AEM.02033-15. Epub 2015 Aug 7.

Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method.

Author information

1
Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Synthetic Biology, University of Groningen, Groningen, The Netherlands.
2
Molecular Genetics Group, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Synthetic Biology, University of Groningen, Groningen, The Netherlands j.w.veening@rug.nl.

Abstract

During the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogen Streptococcus pneumoniae and other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) in S. pneumoniae. Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP in S. pneumoniae and is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling in S. pneumoniae and related organisms.

PMID:
26253684
PMCID:
PMC4579452
DOI:
10.1128/AEM.02033-15
[Indexed for MEDLINE]
Free PMC Article

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