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In vivo selection of sfGFP variants with improved and reliable functionality in industrially important thermophilic bacteria

Overview of attention for article published in Biotechnology for Biofuels, January 2018
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Title
In vivo selection of sfGFP variants with improved and reliable functionality in industrially important thermophilic bacteria
Published in
Biotechnology for Biofuels, January 2018
DOI 10.1186/s13068-017-1008-5
Pubmed ID
Authors

Elrike Frenzel, Jelmer Legebeke, Atze van Stralen, Richard van Kranenburg, Oscar P. Kuipers

Abstract

Fluorescent reporter proteins (FP) have become an indispensable tool for the optimization of microbial cell factories and in synthetic biology per se. The applicability of the currently available FPs is, however, constrained by species-dependent performance and misfolding at elevated temperatures. To obtain functional reporters for thermophilic, biotechnologically important bacteria such as Parageobacillus thermoglucosidasius, an in vivo screening approach based on a mutational library of superfolder GFP was applied. Flow cytometry-based benchmarking of a set of GFPs, sfGFPs and species-specific codon-optimized variants revealed that none of the proteins was satisfyingly detectable in P. thermoglucosidasius at its optimal growth temperature of 60 °C. An undirected mutagenesis approach coupled to fluorescence-activated cell sorting allowed the isolation of sfGFP variants that were extremely well expressed in the chassis background at 60 °C. Notably, a few nucleotide substitutions, including silent mutations, significantly improved the functionality and brightness. The best mutant sfGFP(N39D/A179A) showed an 885-fold enhanced mean fluorescence intensity (MFI) at 60 °C and is the most reliable reporter protein with respect to cell-to-cell variation and signal intensity reported so far. The in vitro spectral and thermostability properties were unaltered as compared to the parental sfGFP protein, strongly indicating that the combination of the amino acid exchange and an altered translation or folding speed, or protection from degradation, contribute to the strongly improved in vivo performance. Furthermore, sfGFP(N39D/A179A) and the newly developed cyan and yellow derivatives were successfully used for labeling several industrially relevant thermophilic bacilli, thus proving their broad applicability. This study illustrates the power of in vivo isolation of thermostable proteins to obtain reporters for highly efficient fluorescence labeling. Successful expression in a variety of thermophilic bacteria proved that the novel FPs are highly suitable for imaging and flow cytometry-based studies. This enables a reliable cell tracking and single-cell-based real-time monitoring of biological processes that are of industrial and biotechnological interest.

Twitter Demographics

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Mendeley readers

The data shown below were compiled from readership statistics for 53 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
Unknown 53 100%

Demographic breakdown

Readers by professional status Count As %
Researcher 12 23%
Student > Ph. D. Student 9 17%
Student > Bachelor 8 15%
Student > Master 7 13%
Other 3 6%
Other 5 9%
Unknown 9 17%
Readers by discipline Count As %
Biochemistry, Genetics and Molecular Biology 25 47%
Agricultural and Biological Sciences 6 11%
Chemistry 3 6%
Immunology and Microbiology 2 4%
Chemical Engineering 2 4%
Other 4 8%
Unknown 11 21%

Attention Score in Context

This research output has an Altmetric Attention Score of 1. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 20 January 2018.
All research outputs
#11,011,140
of 12,390,159 outputs
Outputs from Biotechnology for Biofuels
#812
of 962 outputs
Outputs of similar age
#285,954
of 339,190 outputs
Outputs of similar age from Biotechnology for Biofuels
#18
of 23 outputs
Altmetric has tracked 12,390,159 research outputs across all sources so far. This one is in the 1st percentile – i.e., 1% of other outputs scored the same or lower than it.
So far Altmetric has tracked 962 research outputs from this source. They receive a mean Attention Score of 4.4. This one is in the 1st percentile – i.e., 1% of its peers scored the same or lower than it.
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We're also able to compare this research output to 23 others from the same source and published within six weeks on either side of this one. This one is in the 1st percentile – i.e., 1% of its contemporaries scored the same or lower than it.