Yeast Transformation by the LiAc/SS Carrier DNA/PEG Method

Gietz, R. Daniel; Woods, Robin A.

doi:10.1385/1-59259-958-3:107

Cited by 389 publications

(347 citation statements)

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“…Plasmids were isolated from E. coli cultures using the GeneElute plasmid Miniprep kit (Sigma–Aldrich). The chimeric HXT11/2 and HXT2 expression construct were transformed into S. cerevisiae DS68625 using standard yeast genetic techniques (Gietz and Woods, 2006). …”

Section: Methodsmentioning

confidence: 99%

The amino‐terminal tail of Hxt11 confers membrane stability to the Hxt2 sugar transporter and improves xylose fermentation in the presence of acetic acid

Shin

Nijland

Waal

et al. 2017

Biotech & Bioengineering

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Hxt2 is a glucose repressed, high affinity glucose transporter of the yeast Saccharomyces cerevisiae and is subjected to high glucose induced degradation. Hxt11 is a sugar transporter that is stably expressed at the membrane irrespective the sugar concentration. To transfer this property to Hxt2, the N‐terminal tail of Hxt2 was replaced by the corresponding region of Hxt11 yielding a chimeric Hxt11/2 transporter. This resulted in the stable expression of Hxt2 at the membrane and improved the growth on 8% d‐glucose and 4% d‐xylose. Mutation of N361 of Hxt11/2 into threonine reversed the specificity for d‐xylose over d‐glucose with high d‐xylose transport rates. This mutant supported efficient sugar fermentation of both d‐glucose and d‐xylose at industrially relevant sugar concentrations even in the presence of the inhibitor acetic acid which is normally present in lignocellulosic hydrolysates. Biotechnol. Bioeng. 2017;114: 1937–1945. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

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Section: Methodsmentioning

confidence: 99%

The amino‐terminal tail of Hxt11 confers membrane stability to the Hxt2 sugar transporter and improves xylose fermentation in the presence of acetic acid

Shin

Nijland

Waal

et al. 2017

Biotech & Bioengineering

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“…The resulting fusion constructs in pXN22_GW and pMetYC_GW were transformed into yeast mating strain THY.AP5 (MATα leu2-3, 112 trp1-289 his3-Δ1 loxP::ade2 URA3) and THY.AP4 (MATa leu2-3, 112 ura3-52 lexA::HIS3 lexA::ADE2 lexA::lacZ::trp1) respectively using a modified lithium acetate method. 42 Transformants of AP4 and AP5 expressing Cub and Nub constructs, respectively, were mated by mixing 10µL of each cell suspension in micro-titer plates then spotted on YPAD media and allowed to grow at 28 °C. Once visible growth was observed (overnight to 20 h) cells were scraped and re-suspended in water, and each suspension was spotted on SC -leu -trp +ade +his +met for selection of mated yeasts.…”

Section: Methodsmentioning

confidence: 99%

Inter-subunit interactions between Glutamate-Like Receptors inArabidopsis

Price

Kong

Okumoto

2013

Plant Signaling & Behavior

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“…Clones containing the BD:MYB72, AD:MYB72, BD:EIL3, and AD:EIL3 fusions were checked by sequence analysis and subsequently used in the yeast two-hybrid assay. AD and BD plasmids were transformed into the a and a mating types of Saccharomyces cerevisiae strain PJ69-4, using a lithium acetate/polyethylene glycol protocol described by Gietz and Woods (2006). PJ69-4 carries ADE2, HIS3, URA, and LacZ reporters for reconstituted GAL4 activity (James et al, 1996).…”

Section: Yeast Two-hybrid Assaysmentioning

confidence: 99%

MYB72 Is Required in Early Signaling Steps of Rhizobacteria-Induced Systemic Resistance in Arabidopsis

et al. 2008

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Colonization of Arabidopsis thaliana roots by nonpathogenic Pseudomonas fluorescens WCS417r bacteria triggers a jasmonate/ ethylene-dependent induced systemic resistance (ISR) that is effective against a broad range of pathogens. Microarray analysis revealed that the R2R3-MYB-like transcription factor gene MYB72 is specifically activated in the roots upon colonization by WCS417r. Here, we show that T-DNA knockout mutants myb72-1 and myb72-2 are incapable of mounting ISR against the pathogens Pseudomonas syringae pv tomato, Hyaloperonospora parasitica, Alternaria brassicicola, and Botrytis cinerea, indicating that MYB72 is essential to establish broad-spectrum ISR. Overexpression of MYB72 did not result in enhanced resistance against any of the pathogens tested, demonstrating that MYB72 is not sufficient for the expression of ISR. Yeast two-hybrid analysis revealed that MYB72 physically interacts in vitro with the ETHYLENE INSENSITIVE3 (EIN3)-LIKE3 transcription factor EIL3, linking MYB72 function to the ethylene response pathway. However, WCS417r activated MYB72 in ISR-deficient, ethyleneinsensitive ein2-1 plants. Moreover, exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylate induced wild-type levels of resistance in myb72-1, suggesting that MYB72 acts upstream of ethylene in the ISR pathway. Collectively, this study identified the transcriptional regulator MYB72 as a novel ISR signaling component that is required in the roots during early signaling steps of rhizobacteria-mediated ISR.

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Yeast Transformation by the LiAc/SS Carrier DNA/PEG Method

Cited by 389 publications

References 0 publications

The amino‐terminal tail of Hxt11 confers membrane stability to the Hxt2 sugar transporter and improves xylose fermentation in the presence of acetic acid

The amino‐terminal tail of Hxt11 confers membrane stability to the Hxt2 sugar transporter and improves xylose fermentation in the presence of acetic acid

Inter-subunit interactions between Glutamate-Like Receptors inArabidopsis

MYB72 Is Required in Early Signaling Steps of Rhizobacteria-Induced Systemic Resistance in Arabidopsis

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