Enhancement of plasmid DNA transformation efficiencies in early stationary‐phase yeast cell cultures

Tripp, Jennifer DeMars; Lilley, Jennifer L.; Wood, Whitney N.; Lewis, L. Kevin

doi:10.1002/yea.2951

Cited by 33 publications

(20 citation statements)

References 36 publications

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“…The ELO3 coding sequence was deleted using homologous recombination by amplifying the hphMX6 gene conferring hygromycin resistance from the vector pAG32 (Addgene) using the primers elo3F and elo3R (IDT). The PCR product designed for homologous recombination was transformed using lithium acetate (Sigma) as previously described (Tripp et al, 2013), and cells were grown on YPD agar (RPI) plates containing 200 mg/L hygromycin B (Goldbio). The high expression dsRed-Rho1 vector was created using the splicing by overlap extension method (Higuchi et al, 1988).…”

Section: Yeast Strains and Growth Conditionsmentioning

confidence: 99%

Sphingolipids regulate the tethering stage of vacuole fusion by affecting membrane fluidity

Hurst

Zhang

Kazmirchuk

et al. 2020

Preprint

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ORCIDRutilio Fratti -https://orcid.org/0000-0001-9109-6666 Running title: Elo3p regulation of vacuole fusion via lipid rafts SummaryMany sphingolipids contain very-long chain fatty-acids with 26 carbons. The deletion of Elo3, the elongase that adds the final two carbons results in pleiotropic effects that negatively alter membrane fusion at the tethering and docking stages. AbstractThe role of sphingolipids in controlling the endolysosomal membrane trafficking remains unclear. Here, we show that in Saccharomyces cerevisiae sphingolipids containing very long-chain fatty-acids (VLCAs) promote homotypic vacuolar fusion. Yeast that lack the C26 VLCA elongase Elo3p display morphological and vacuolar abnormalities. Vacuoles isolated from these cells displayed reduced levels of in vitro fusion, which we traced to a block in tethering and docking. We found that C26 VLCFA deficient yeast mislocalize fusion markers, and the small GTPases Rho1p and Ypt7p fail to selectively concentrate at the boundary and vertex domains of vacuoles isolated from these yeasts. Surprisingly, we only observed mild changes to the localization of other regulatory lipids, but membrane fluidity and solubility was significantly altered. Taken together, these results suggest that sphingolipids containing C26 VLCFAs act as regulatory lipids in the homotypic vacuolar fusion cascade by assembling membrane microdomains that promote the protein and lipid machinery required for the tethering and docking of vacuoles.

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Section: Yeast Strains and Growth Conditionsmentioning

confidence: 99%

Sphingolipids regulate the tethering stage of vacuole fusion by affecting membrane fluidity

Hurst

Zhang

Kazmirchuk

et al. 2020

Preprint

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“…Transformation of Saccharomyces cerevisiae yeast strains was performed according to Tripp et al . (2013) utilizing an optimized LiAc-method. Prey plasmids were transformed into Y187 strain and bait plasmids were transformed into AH109 strain.…”

Section: Methodsmentioning

confidence: 99%

Towards deorphanizing G protein-coupled receptors of Schistosoma mansoni using the MALAR yeast two-hybrid system

et al. 2019

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Schistosomiasis is an acute and chronic disease caused by parasitic worms of the genus Schistosoma. Treatment is solely dependent on praziquantel. In the face of the worldwide dimension, projects have been initiated to develop new chemotherapies. Due to their proven druggability, G protein-coupled receptors (GPCRs) are promising targets for anthelmintics. However, to identify candidate receptors, a deeper understanding of GPCR signalling in schistosome biology is essential. Comparative transcriptomics of paired and unpaired worms and their gonads revealed 59 differentially regulated GPCR-coding genes putatively involved in neuronal processes. In general, the diversity among GPCRs and their integral membrane topology make it difficult to characterize and deorphanize these receptors. To overcome existing limitations, we performed a pilot approach and utilized the innovative Membrane-Anchored Ligand And Receptor yeast two-hybrid system (MALAR-Y2H) to associate potential neuropeptide ligands with their cognate receptors. Here, we demonstrated the ability to express full-length GPCRs of Schistosoma mansoni in a heterologous yeast-based system. Additionally, we localized GPCRs and chimeras of neuropeptides fused to the WBP1 transmembrane domain of yeast to the plasma membrane of yeast cells. Reporter gene assays indicated ligand-receptor binding, which allowed us to identify certain neuropeptides as potential ligands for two GPCRs, which had been found before to be differentially expressed in schistosomes in a pairing-dependent manner. Thus, the MALAR-Y2H system appears suitable to unravel schistosome GPCR–ligand interactions. Besides its relevance for understanding schistosome biology, identifying and characterizing GPCR–ligand interaction will also contribute to applied research aspects.

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“…The eight parent strains that were selected based on pre-screenings were transformed with CRISPR/Cas9 plasmids containing protospacer sequences targeting either MATa and MATα using optimized stationary phase transformation (Tripp et al 2013). Transformation efficiencies varied broadly, with between 1 and 133 colonies emerging on the selection plates (400 mg hygromycin / mL) from the transformation of 1.5 mL saturated overnight culture (Table 1).…”

Section: Generating Mating-competent Variants For Hybridizationmentioning

confidence: 99%

Efficient breeding of industrial brewing yeast strains using CRISPR/Cas9-aided mating-type switching

Krogerus

Fletcher²,

Rettberg

et al. 2021

Preprint

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Yeast breeding is a powerful tool for developing and improving brewing yeast in a number of industry-relevant respects. However, breeding of industrial brewing yeast can be challenging, as strains are typically sterile and have large complex genomes. To facilitate breeding, we used the CRISPR/Cas9 system to generate double-stranded breaks in the MAT locus, generating transformants with a single specified mating type. The single mating type remained stable even after loss of the Cas9 plasmid, despite the strains being homothallic, and these strains could be readily mated with other brewing yeast transformants of opposite mating type. As a proof of concept, we applied this technology to generate yeast hybrids with an aim to increase beta-lyase activity for fermentation of beer with enhanced hop flavour. First, a genetic and phenotypic pre-screening of 38 strains was carried out in order to identify potential parent strains with high beta-lyase activity. Mating-competent transformants of eight parent strains were generated, and these were used to generate over 60 hybrids that were screened for beta-lyase activity. Selected phenolic off-flavour positive (POF+) hybrids were further sporulated to generate meiotic segregants with high beta-lyase activity, efficient wort fermentation and lack of POF; all traits that are desirable in strains for the fermentation of modern hop-forward beers. Our study demonstrates the power of combining the CRISPR/Cas9 system with classic yeast breeding to facilitate development and diversification of brewing yeast.

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Enhancement of plasmid DNA transformation efficiencies in early stationary‐phase yeast cell cultures

Cited by 33 publications

References 36 publications

Sphingolipids regulate the tethering stage of vacuole fusion by affecting membrane fluidity

Sphingolipids regulate the tethering stage of vacuole fusion by affecting membrane fluidity

Towards deorphanizing G protein-coupled receptors of Schistosoma mansoni using the MALAR yeast two-hybrid system

Efficient breeding of industrial brewing yeast strains using CRISPR/Cas9-aided mating-type switching

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