<i>In vivo</i> characterisation of fluorescent proteins in budding yeast

Botman, Dennis; Groot, Daan H. de; Schmidt, Phillipp; Goedhart, Joachim; Teusink, Bas

doi:10.1101/431874

Cited by 12 publications

(24 citation statements)

References 59 publications

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“…The new yellow-green fluorescent protein has been used mainly for fluorescent microscopy. It has been applied as genetically encoded tag to visualize gene products and cellular compartments in bacteria, animals, plants and yeast (Botman et al 2019;Heppert et al 2016;Hostettler et al 2017;Stoddard and Rolland 2019;Wilton et al 2017). Only one report exists on expression mNG in filamentous fungi.…”

Section: Introductionmentioning

confidence: 99%

Establishment of the monomeric yellow-green fluorescent protein mNeonGreen for life cell imaging in mycelial fungi

et al. 2020

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The engineered monomeric version of the lancelet Branchiostoma lanceolatum fluorescent protein, mNeonGreen (mNG), has several positive characteristics, such as a very bright fluorescence, high photostability and fast maturation. These features make it a good candidate for the utilization as fluorescent tool for cell biology and biochemical applications in filamentous fungi. We report the generation of plasmids for the expression of the heterologous mNG gene under the control of an inducible and a constitutive promoter in the filamentous ascomycete Sordaria macrospora and display a stable expression of mNG in the cytoplasm. To demonstrate its usefulness for labeling of organelles, the peroxisomal targeting sequence serine-lysine-leucine (SKL) was fused to mNG. Expression of this tagged version led to protein import of mNG into peroxisomes and their bright fluorescence in life cell imaging.

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Section: Introductionmentioning

confidence: 99%

Establishment of the monomeric yellow-green fluorescent protein mNeonGreen for life cell imaging in mycelial fungi

et al. 2020

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“…When the coding sequence of MRX12 was replaced with a novel ORF (i.e. LEU2 or deCFP), the new coding sequence was consistently coregulated with Figure 22) (Botman et al 2019). In our study, we determined that the intensity of the fluorophores was not the issue: we could detect each of the reporters in our control strains.…”

Section: Development Of a Dual-reporter System For Studying Agcmentioning

confidence: 71%

“…Furthermore, these two fluorophores are more vibrant and photostable as compared to traditional GFPs and RFPs (i.e. mCherry) (Lee et al 2013, Botman et al 2019; photostability should protect against loss of protein signal due to photobleaching. Additionally, yemVenus folds faster than its counterpart GFP, making it more reflective of variable gene transcription (Giuraniuc et al 2013).…”

Section: Section Iv: Fluorescent Reporters Can Be Used For Measuring mentioning

confidence: 99%

“…Both mRuby2 and eCFP under the control of pMRX12 on a plasmid system were transcribed appropriately but appear to not be translated. In order to address this problem, we tried yet another reporter and acquired a plasmid that expressed a RFP and a BFP well in S. cerevisiae (Botman et al 2019). First, we generated a fragment for the RFP mKate2 and cloned it under pMRX12 with a degron-tag.…”

Section: Figure 24 -Yemvenus But Not Ecfp Is Repressed At the Levelmentioning

confidence: 99%

“…Recent literature indicates that while mRuby2 can be expressed in S. cerevisiae, other fluorophores may be better candidates for this species. An extensive characterization of fluorophores in S. cerevisiae found that mRuby2 fluoresces with "relatively low brightness", on the other hand, proteins like eCFP, mTq2, mKate2 and tdTomato fluoresced with "high brightness"(Figure 22)(Botman et al 2019). Since in the past, we have successfully visualized BFPs, we replaced mRuby2 with eCFP under the control of pMRX12.…”

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confidence: 99%

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Developing And Testing A Two-color Assay For Measuring Adjacent Gene Coregulation In S. Cerevisiae

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Organisms regulate gene expression at the transcriptional level to meet the variable needs of the cell. These needs change in response to environmental stimuli and developmental needs, and one of the biggest and most complex pathways that microbial cells need to control is that of ribosome biosynthesis. Ribosomal biogenesis in yeast requires hundreds of gene products to produce upwards of 2,000 ribosomes per minute. This tightly regulated metabolic process depends on the synthesis of rRNAs and ribosomal proteins, in conjunction with a coregulated set of genes constituting the rRNA and ribosomes biogenesis regulon (RRB). The RRB regulon of S. cerevisiae consists of over 200 genes including those that process and assemble the ribosomes. Interestingly, a significant fraction of these RRB genes (28) occur as adjacent pairs on the chromosome. Gene expression analysis of the coregulated MPP10-MRX12 RRB gene pair has shown that the promoter of MPP10 regulates the transcription rate of MRX12. This phenomenon has come to be known as adjacent gene coregulation (AGC). Typically, mRNA expression is monitored through qRT-PCR analysis. However, this technique can be laborious and a visual assay for measuring gene expression would prove to be a useful alternative. Here we investigate the effectiveness of a two-color assay for monitoring AGC. Through Gibson assembly, we created a plasmid that replaced the adjacent gene pair of MPP10-MRX12 with yemVenus-mRuby2 open reading frames. Fluorescent microscopy and flow cytometry were then utilized to analyze the fluorescent intensity produced by yemVenus (green) and mRuby2 (red), and this was used as a proxy for mRNA expression. The strain carrying the reporter plasmid was subjected to heat shock and the mRNA levels of both reporters were repressed similar to the expression of the chromosomal MPP10-MRX12. However, only yemVenus produced a fluorescent signal that was visible above background, and it was repressed throughout a heat shock. We then screened through five alternative fluorescent genes to replace mRuby2, all of which were repressed transcriptionally, but did not produce a visible fluorescent signal. In tandem, our reporter system was transformed into various trans-and cis-factor mutated strains known to disrupt AGC. However, these factors only abrogate AGC on the chromosome and do not affect the co-repression of adjacent genes on the plasmid. These results suggest that proper AGC regulation may depend on structure that is unique to the chromosomal context. Ultimately, we may integrate these reporters into the chromosome in order to shed more light on the mechanisms behind the regulation of paired genes in coregulated metabolic pathways.

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Rapid directed molecular evolution of fluorescent proteins in mammalian cells

et al. 2021

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In vivo imaging of model organisms is heavily reliant on uorescent proteins with high intracellular brightness. Here we describe a practical method for rapid optimization of uorescent proteins via directed molecular evolution in cultured mammalian cells. Using this method, we were able to perform screening of large gene libraries containing up to 2•10 7 independent random genes of uorescent proteins expressed in HEK cells completing one iteration directed evolution in a course of ~ 8 days. We employed this approach to develop a set of green and near-infrared uorescent proteins with enhanced intracellular brightness. The developed near-infrared uorescent proteins demonstrated high performance for uorescent labeling of neurons in culture and in vivo in model organisms such as C.elegans, Drosophila, zebra sh, and mice. Spectral properties of the optimized near-infrared uorescent proteins enabled crosstalk-free multicolor imaging in combination with common green and red uorescent proteins, as well as dual-color near-infrared uorescence imaging. The described method has a great potential to be adopted by protein engineers due to its simplicity and practicality. We also believe that the new enhanced uorescent proteins will nd wide application for in vivo multi-color imaging of small model organisms.

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In vivo characterisation of fluorescent proteins in budding yeast

Cited by 12 publications

References 59 publications

Establishment of the monomeric yellow-green fluorescent protein mNeonGreen for life cell imaging in mycelial fungi

Establishment of the monomeric yellow-green fluorescent protein mNeonGreen for life cell imaging in mycelial fungi

Developing And Testing A Two-color Assay For Measuring Adjacent Gene Coregulation In S. Cerevisiae

Rapid directed molecular evolution of fluorescent proteins in mammalian cells

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