In vivo characterisation of fluorescent proteins in budding yeast

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

Fluorescent proteins (FPs) are widely used in many organisms, but are commonly characterised in vitro. However, the in vitro properties may poorly reflect in vivo performance. Therefore, we characterised 27 FPs in vivo using Saccharomyces cerevisiae as model organism. We linked the FPs via a T2A peptide to a control FP, producing equimolar expression of the 2 FPs from 1 plasmid. Using this strategy, we characterised the FPs for brightness, photostability, photochromicity and pH-sensitivity, achieving a comprehensive in vivo characterisation. Many FPs showed different in vivo properties compared to existing in vitro data. Additionally, various FPs were photochromic, which affects readouts due to complex bleaching kinetics. Finally, we codon optimized the best performing FPs for optimal expression in yeast, and found that codon-optimization alters FP characteristics. These FPs improve experimental signal readout, opening new experimental possibilities. Our results may guide future studies in yeast that employ fluorescent proteins.

Original languageEnglish
Article number2234
Pages (from-to)1-14
Number of pages14
JournalScientific Reports
Volume9
DOIs
Publication statusPublished - 19 Feb 2019

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Saccharomycetales
Proteins
Codon
Yeasts
Saccharomyces cerevisiae
Plasmids

Cite this

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title = "In vivo characterisation of fluorescent proteins in budding yeast",
abstract = "Fluorescent proteins (FPs) are widely used in many organisms, but are commonly characterised in vitro. However, the in vitro properties may poorly reflect in vivo performance. Therefore, we characterised 27 FPs in vivo using Saccharomyces cerevisiae as model organism. We linked the FPs via a T2A peptide to a control FP, producing equimolar expression of the 2 FPs from 1 plasmid. Using this strategy, we characterised the FPs for brightness, photostability, photochromicity and pH-sensitivity, achieving a comprehensive in vivo characterisation. Many FPs showed different in vivo properties compared to existing in vitro data. Additionally, various FPs were photochromic, which affects readouts due to complex bleaching kinetics. Finally, we codon optimized the best performing FPs for optimal expression in yeast, and found that codon-optimization alters FP characteristics. These FPs improve experimental signal readout, opening new experimental possibilities. Our results may guide future studies in yeast that employ fluorescent proteins.",
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In vivo characterisation of fluorescent proteins in budding yeast. / Botman, Dennis; de Groot, Daan Hugo; Schmidt, Phillipp; Goedhart, Joachim; Teusink, Bas.

In: Scientific Reports, Vol. 9, 2234, 19.02.2019, p. 1-14.

Research output: Contribution to JournalArticleAcademicpeer-review

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AU - Botman, Dennis

AU - de Groot, Daan Hugo

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