Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method

Gietz, R. Daniel; Schiestl, Robert H.

doi:10.1038/nprot.2007.14

Cited by 390 publications

(296 citation statements)

References 6 publications

Supporting

Mentioning

286

Contrasting

Unclassified

Order By: Relevance

“…Escherichia coli DH10B and plasmid pJET1.2 (Fermentas) were used for routine cloning and sequencing. S. cerevisiae BJ5464-NpgA (MATα ura3-52 his3-Δ200 leu2-Δ1 trp1 pep4::HIS3 prb1 Δ1.6R can1 GAL) (34, 41) was maintained on yeast extract peptone dextrose agar (Difco) and transformed using the small-scale lithium chloride protocol (42). The yeast-E. coli shuttle vectors YEpADH2p-FLAG-URA and YEpADH2p-FLAG-TRP (20) are based on the YEpADH2p vectors with the URA3 or the TRP1 selectable markers (13).…”

Section: Methodsmentioning

confidence: 99%

Rational reprogramming of fungal polyketide first-ring cyclization

Zhou

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

115

View full text Add to dashboard Cite

Resorcylic acid lactones and dihydroxyphenylacetic acid lactones represent important pharmacophores with heat shock response and immune system modulatory activities. The biosynthesis of these fungal polyketides involves a pair of collaborating iterative polyketide synthases (iPKSs): a highly reducing iPKS with product that is further elaborated by a nonreducing iPKS (nrPKS) to yield a 1,3-benzenediol moiety bridged by a macrolactone. Biosynthesis of unreduced polyketides requires the sequestration and programmed cyclization of highly reactive poly-β-ketoacyl intermediates to channel these uncommitted, pluripotent substrates to defined subsets of the polyketide structural space. Catalyzed by product template (PT) domains of the fungal nrPKSs and discrete aromatase/cyclase enzymes in bacteria, regiospecific first-ring aldol cyclizations result in characteristically different polyketide folding modes. However, a few fungal polyketides, including the dihydroxyphenylacetic acid lactone dehydrocurvularin, derive from a folding event that is analogous to the bacterial folding mode. The structural basis of such a drastic difference in the way a PT domain acts has not been investigated until now. We report here that the fungal vs. bacterial folding mode difference is portable on creating hybrid enzymes, and we structurally characterize the resulting unnatural products. Using structure-guided active site engineering, we unravel structural contributions to regiospecific aldol condensations and show that reshaping the cyclization chamber of a PT domain by only three selected point mutations is sufficient to reprogram the dehydrocurvularin nrPKS to produce polyketides with a fungal fold. Such rational control of first-ring cyclizations will facilitate efforts to the engineered biosynthesis of novel chemical diversity from natural unreduced polyketides.

show abstract

Section: Methodsmentioning

confidence: 99%

Rational reprogramming of fungal polyketide first-ring cyclization

Zhou

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

115

View full text Add to dashboard Cite

show abstract

“…Strains created for this study were constructed using heterologous gene replacement (33,34) or through classical yeast mating in combination with tetrad dissection or high throughput strain construction techniques (35). Set2 expression plasmids (14,21) were transformed using the lithium chloride technique (36), and shuffle strains (where important genes were genomically deleted but carried on a uracil-selectable plasmid) were maintained on selective media until the time of the appropriate experiment, and plasmid loss was instigated by plating on media containing 5-fluoroorotic acid (5-FOA) (37).…”

Section: Methodsmentioning

confidence: 99%

RNA Polymerase II Carboxyl-terminal Domain Phosphorylation Regulates Protein Stability of the Set2 Methyltransferase and Histone H3 Di- and Trimethylation at Lysine 36

Fuchs

Kizer

Braberg

et al. 2012

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: Set2 catalyzes mono-, di-, and trimethylation of lysine 36 on histone H3 (H3K36). Results: Phosphorylation of the C-terminal domain of RNA polymerase II (RNAPII CTD) regulates Set2 protein levels and H3K36 methylation. Conclusion: Set2 protein is regulated to maintain balanced levels of H3K36 methylation. Significance: These results suggest that different methylation states perform distinct biological functions.

show abstract

“…All assembled cassette parts which were amplified by PCR (pUC13 forward and reverse primers) and inserted into pYES2 plasmid were transformed into S. cerevisiae competent cells following standard method in combination with Invitrogen kit protocol (Invitrogen Inc., CA) [25,26]. However, the Invitrogen Inc. protocol was preferred in the denoted investigation as follows:…”

Section: Transformation Of Yeast Saccharomyces Cerevisiae (Sc)mentioning

confidence: 99%

Construct of DNA glucose sensor yeast plasmid for early detection of diabetes

Cuero¹,

Navia²,

Agudelo³

et al. 2017

Integr Obesity Diabetes

View full text Add to dashboard Cite

This investigation was aimed at assembling different genetic building blocks to produce a focused DNA sensor to detect proteins related to glucose in blood, in order to diagnose early diabetes using synthetic biology and conventional molecular biology. A glucose DNA sensor was constructed in Saccharomyces cerevisiae using genomic and synthesized sequences and were tested in both, In vivo using human blood plasma sampled, and In vitro using cultural media. The results were based on fluorescence intensity of the DNA sensor and compared with clinical methods. The DNA sensor was highly sensitive when mixed with plasma samples from different patients (i.e., diabetic, pre-diabetic, and normal) showing high fluorescence, and was able to detect a wide concentration range of glucose equivalent to clinical glycaemia values. Expression of proteins pertaining to glucose metabolism production was determined by 2D-DIGE gel electrophoresis-maldi analysis. The glucose sensor produced results less than a minute after being mixed with a drop of a human blood sample. Our results highlight the advantages of using constructed DNA sensors to detect glucose in blood for early diagnosis of diabetes.The yeast DNA glucose sensor was assembled successfully with standardized genetic parts and was able to detect low levels equivalent to clinical glycaemia (<140 mg/ dL), as well as a higher equivalent level of glycaemia (>200 mg/dL). Thus, our sensor can be used for early diagnosis of diabetes or pre-diabetic conditions, thereby allowing for earlier clinical intervention. The direct correlation between levels of glucose and the intensity of fluorescence of the DNA sensor shows the advantage of using this technology in order to identify specific types of diabetic patients. Sometimes, it is difficult to establish accurate correlations with different methods.human blood [7]. These types of sensors are versatile analytical tools that offer advantages over classical analytical methods due to their inherent specificity, selectivity, and simplicity [5]. Most biological and cellular sensors use membrane and cellular proteins that recognize and take up biomarkers from a medium [6]. However, DNA has also been used in molecular biosensors [8,9].Biomarkers for detecting and diagnosing diseases include physical symptoms, mutated DNA and RNA, secreted proteins, processes such as cell death or proliferation, and serum concentrations of small molecules such as cholesterol or, in the case of diabetes, glucose [6].Currently, the most common methods to diagnose diabetes use enzymatic reactions to determine glucose levels [10,11]. These methods measure glucose in whole blood because erythrocytes contain high concentrations of glucose [12]. The methods include the use of glucose oxidase, hexokinase, and glucose dehydrogenase [13][14][15]. The products of these reactions with blood sugar can be determined using colorimetric and spectrophotometric assays or by measuring the electric current produced in the enzyme reactions; the latter is the approach used ...

show abstract

Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method

Cited by 390 publications

References 6 publications

Rational reprogramming of fungal polyketide first-ring cyclization

Rational reprogramming of fungal polyketide first-ring cyclization

RNA Polymerase II Carboxyl-terminal Domain Phosphorylation Regulates Protein Stability of the Set2 Methyltransferase and Histone H3 Di- and Trimethylation at Lysine 36

Construct of DNA glucose sensor yeast plasmid for early detection of diabetes

Contact Info

Product

Resources

About