Journal: Microbial Cell Factories

Article Title: Dynamic regulation of gene expression using sucrose responsive promoters and RNA interference in Saccharomyces cerevisiae

doi: 10.1186/s12934-015-0223-7

Figure Lengend Snippet: Testing promoters for differential expression on sucrose. SUC2, GLC3, MAL12 , and GPH1 promoters were used to control GFP expression. Each strain was grown to mid-exponential phase on minimal medium containing 10 g/L glucose or 10 g/L sucrose prior to flow cytometry-based GFP measurement. Mean GFP fluorescence levels in arbitrary units (au) from duplicate fermentations with error bars representing the standard deviation are shown.

Article Snippet: Plasmids P TEF1 -yEGFPCLN2PEST-pRS406, P SUC2 -yEGFPCLN2PEST-pRS406, and P SUC2 -GFPantisense-CYC1t-pRS413 will be made available from AddGene ( www.addgene.org/ ).

Techniques: Quantitative Proteomics, Control, Expressing, Flow Cytometry, Fluorescence, Standard Deviation

Journal: Microbial Cell Factories

Article Title: Dynamic regulation of gene expression using sucrose responsive promoters and RNA interference in Saccharomyces cerevisiae

doi: 10.1186/s12934-015-0223-7

Figure Lengend Snippet: Tuning the timing of gene expression using different ratios of glucose to sucrose. A strain expressing the destabilized GFP gene driven by the SUC2 promoter was grown in media containing the indicated concentrations of glucose and sucrose. Extracellular glucose, sucrose, and fructose concentrations were measured using HPLC during the initial growth phase alongside GFP expression levels. Population density (OD 660nm ) and GFP expression levels were measured up to 56 hours of shake-flask cultivation. All data points and error bars represent the mean and standard deviation from triplicate cultivations.

Article Snippet: Plasmids P TEF1 -yEGFPCLN2PEST-pRS406, P SUC2 -yEGFPCLN2PEST-pRS406, and P SUC2 -GFPantisense-CYC1t-pRS413 will be made available from AddGene ( www.addgene.org/ ).

Techniques: Gene Expression, Expressing, Standard Deviation

Journal: Microbial Cell Factories

Article Title: Dynamic regulation of gene expression using sucrose responsive promoters and RNA interference in Saccharomyces cerevisiae

doi: 10.1186/s12934-015-0223-7

Figure Lengend Snippet: Comparison of TEF1 and SUC2 promoter strengths. GFP fluorescence (a) and population density (b) were measured for P TEF1 - GFP and P SUC2 - GFP expressing strains in 1% glucose, 1% sucrose containing medium over 48 hours. Mean and standard deviation for triplicate cultivations are shown.

Article Snippet: Plasmids P TEF1 -yEGFPCLN2PEST-pRS406, P SUC2 -yEGFPCLN2PEST-pRS406, and P SUC2 -GFPantisense-CYC1t-pRS413 will be made available from AddGene ( www.addgene.org/ ).

Techniques: Comparison, Fluorescence, Expressing, Standard Deviation

Journal: Microbial Cell Factories

Article Title: Dynamic regulation of gene expression using sucrose responsive promoters and RNA interference in Saccharomyces cerevisiae

doi: 10.1186/s12934-015-0223-7

Figure Lengend Snippet: Dynamic repression of GFP expression using sucrose mediated RNAi. (a) The expression of an antisense RNA results in the destruction of complementary mRNA via Dicer and Argonaute enzymes. (b) Expression of the full GFP ORF in the antisense direction is triggered during growth on sucrose using the SUC2 promoter, causing constitutively regulated ( TEF1 promoter) GFP expression to be repressed via Dicer/Argonaute-mediated RNA interference. GFP expression levels (c) , and population density (d) were measured for P TEF1 - GFP expressing strains both with (‘RNAi’, black triangles) and without (‘control’, green circles) a P SUC2 - GFP antisense construct. The GFP expression level from the control strain was set to ‘100%’, with GFP expression values from the RNAi strain being normalised to this value. Means ± standard deviations are shown from triplicate cultures. Figure 4b was adapted from . Non-normalised GFP expression values are shown in Additional file : Figure S1.

Article Snippet: Plasmids P TEF1 -yEGFPCLN2PEST-pRS406, P SUC2 -yEGFPCLN2PEST-pRS406, and P SUC2 -GFPantisense-CYC1t-pRS413 will be made available from AddGene ( www.addgene.org/ ).

Techniques: Expressing, Control, Construct

Journal: Microbial Cell Factories

Article Title: Dynamic regulation of gene expression using sucrose responsive promoters and RNA interference in Saccharomyces cerevisiae

doi: 10.1186/s12934-015-0223-7

Figure Lengend Snippet: S. cerevisiae strains used in this study

Article Snippet: Plasmids P TEF1 -yEGFPCLN2PEST-pRS406, P SUC2 -yEGFPCLN2PEST-pRS406, and P SUC2 -GFPantisense-CYC1t-pRS413 will be made available from AddGene ( www.addgene.org/ ).

Techniques: Expressing, Control

Journal: Microbial Cell Factories

Article Title: Dynamic regulation of gene expression using sucrose responsive promoters and RNA interference in Saccharomyces cerevisiae

doi: 10.1186/s12934-015-0223-7

Figure Lengend Snippet: Plasmids

Article Snippet: Plasmids P TEF1 -yEGFPCLN2PEST-pRS406, P SUC2 -yEGFPCLN2PEST-pRS406, and P SUC2 -GFPantisense-CYC1t-pRS413 will be made available from AddGene ( www.addgene.org/ ).

Techniques: Plasmid Preparation, Expressing, Selection, Marker, Construct

Journal: Journal of Cell Science

Article Title: Modified chromosome structure caused by phosphomimetic H2A modulates the DNA damage response by increasing chromatin mobility in yeast

doi: 10.1242/jcs.258500

Figure Lengend Snippet: Mimicking H2A S129 phosphorylation increases NHEJ and decreases translocation rates. (A) Schematic of the NHEJ assay principle. A replicative plasmid carrying HIS3 as an auxotrophic marker (pRS413) is linearized by EcoRI enzyme (cs, cut site) and used for yeast transformation. In strains with efficient NHEJ, plasmid extremities are joined (arrow) and His+ colonies are recovered on plates lacking histidine. (B) Rejoining efficiency was calculated for the indicated strains as the percentage recovery of His+ colonies after transformation of a linearized plasmid, relative to recovery following introduction of a non-linearized plasmid. A Δyku70 mutant (Ku70) is impaired in NHEJ and was used as a control for plasmid digestion efficiency. The bar graph shows the mean±s.e.m. of five experiments. **P≤0.01; ***P≤0.0001; n.s., not significant (non-parametric t-test). (C) Schematic of the strain used to measure translocation. Two DSBs generated by HO endonuclease (HO cs), an intronic sequence (dotted line) and truncated forms of URA3 are depicted on two distinct chromosomes (Chr. III and Chr. V). Reciprocal end-joining produces Ura+ cells. AI, artificial intron. (D) Percentage translocation efficiency for the indicated strains was calculated as the ratio of the number of Ura+ colonies recovered after persistent induction of DSBs (on −URA plates containing galactose) relative to the number recovered in non-translocated conditions (+URA plates containing galactose). The bar graph shows the mean±s.d. of five experiments. **P≤0.01; ***P≤0.0001; n.s., not significant (non-parametric t-test).

Article Snippet: Plasmid repair assay The plasmid pRS413 ( HIS3 ; Addgene) was linearized with EcoR I, which generates cohesive ends.

Techniques: Phospho-proteomics, Translocation Assay, NHEJ Assay, Plasmid Preparation, Marker, Transformation Assay, Mutagenesis, Control, Generated, Sequencing