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

Détails

ID Serval
serval:BIB_219A8DC8FEFF
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method.
Périodique
Applied and Environmental Microbiology
Auteur⸱e⸱s
Beilharz K., van Raaphorst R., Kjos M., Veening J.W.
ISSN
1098-5336 (Electronic)
ISSN-L
0099-2240
Statut éditorial
Publié
Date de publication
2015
Volume
81
Numéro
20
Pages
7244-7252
Langue
anglais
Résumé
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.
Mots-clé
Green Fluorescent Proteins/genetics, Green Fluorescent Proteins/metabolism, Luminescent Proteins/genetics, Luminescent Proteins/metabolism, Microscopy, Fluorescence, Molecular Sequence Data, Streptococcus pneumoniae/genetics, Streptococcus pneumoniae/metabolism
Pubmed
Web of science
Open Access
Oui
Création de la notice
11/10/2016 16:36
Dernière modification de la notice
20/08/2019 13:58
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