Journal: Molecular cell

Article Title: An isoprene lipid binding protein promotes eukaryotic coenzyme Q biosynthesis

doi: 10.1016/j.molcel.2018.11.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: S. cerevisiae ∆coq9 BY4742 alpha haploid his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 , Thermo collection #YSC1054 , Thermo YSC6272-201920350.

Techniques: Produced, Magnetic Beads, Virus, Recombinant, Staining, Software, Real-time Polymerase Chain Reaction

Journal: Microbial Cell Factories

Article Title: Fine-tuning the expression of pathway gene in yeast using a regulatory library formed by fusing a synthetic minimal promoter with different Kozak variants

doi: 10.1186/s12934-021-01641-z

Figure Lengend Snippet: A general workflow employed in the current study. The chimeric promoter library was generated by randomizing the hexameric Kozak motif of the UAS F-E-C -Core1 artificial promoter to any of the four DNA bases. Several Kozak variants with strong protein expression activity were obtained by GFP fluorescence screening. This library was then applied for metabolic pathway optimization in S. cerevisiae BY4742 strain

Article Snippet: The MATa haploid strain of S. cerevisiae BY4742 (Thermo Fisher Scientific, Waltham, MA, USA) was used as a host for promoter engineering and combinatorial pathway optimization.

Techniques: Generated, Expressing, Activity Assay, Fluorescence

Journal: Microbial Cell Factories

Article Title: Fine-tuning the expression of pathway gene in yeast using a regulatory library formed by fusing a synthetic minimal promoter with different Kozak variants

doi: 10.1186/s12934-021-01641-z

Figure Lengend Snippet: Strains and plasmids used in this work

Article Snippet: The MATa haploid strain of S. cerevisiae BY4742 (Thermo Fisher Scientific, Waltham, MA, USA) was used as a host for promoter engineering and combinatorial pathway optimization.

Techniques: Plasmid Preparation, In Situ, Variant Assay

Journal: bioRxiv

Article Title: Bayesian analysis dissects kinetic modulation during non-stationary gene expression

doi: 10.1101/2023.06.20.545522

Figure Lengend Snippet: a Oscillations in CUP1 mRNA expression level quantified by RT-qPCR and normalized by expression of the housekeeping gene ACT1 . Error bars represent standard error of the mean (SEM) from two biological replicates. b Schematic of the 14x PP7 reporter replacing one copy of the CUP1 ORF in chromosome VIII of S. cerevisiae . Two types of the stem loop sequence (red and green stems, purple loops, or bulges) are present in the reporter sequence, each stem is bound by a PP7-GFP dimer. Hence, 28 GFP molecules associate with single mRNA. The length of reporter transcript is 862 bp, however a few transcripts may be longer as there is no terminator immediately after 14xPP7. The expression of KANMX observed by RT-qPCR is constitutive and does not follow oscillations of 14X PP7 transcripts, which indicates that great majority of 14xPP7 transcripts stop before KANMX ORF (data not shown). c Example field view of cells with active TS containing nascent 14x PP7 reporter transcripts. Cells were imaged after 9 min of Cu 2+ induction. Z-stack of the entire cell volume is presented as a maximum intensity projection for the GFP channel. Scale: 5 μm. d TS in individual cell display independent spikes of activity. TS dynamics from 10 representative cells are presented for the first 21 min since Cu 2+ addition, imaged with 1 min time-lapse. Maximum intensity projections of the entire z-volume of the cells were cropped by keeping the TS in the center of the 13x13 pixels area. e Illustration of the movie collection for datasets imaged with 3 s time-lapse. Movies on the same coverslip are started every 3 min and are recorded for 90 s (green blocks). The remaining time is used to move the microscope to the next position on the coverslip. By collecting several such movie sequences starting either 3 min or 4.5 min after induction, the whole first cycle is covered. f The fraction of cells in the population showing an active TS follows the oscillation pattern of expression of the whole CUP1 locus. Cells with TS were counted in independent fields imaged sequentially with 3 min time interval. Error bars represent standard error of the percentage (SEP). g TS in individual cells express more transcripts at the peak of the oscillation. In sequential 90 s movies collected after Cu 2+ addition with 3 s time-lapse, the spot intensities were measured in the first frame of every movie. The graph shows the population average of these spot intensities indicating that transcriptional output of individual cells follows the oscillation pattern of the whole system. h Schematics of CUP1 multi-scale bursting. Top – long oscillations between transcription (ON1 state) and no-transcription phases (OFF1 state) on the population level (cf. a and d ). Bottom – short transcription spikes (ON2 and OFF2 states) as observed for individual TS ( f ). Transcriptional amplitude of the spikes is defined by the average number of mRNA produced per spike (striped lines represent different nascent mRNA produced during spike). The gene switches from an inactive OFF2 state to an active ON2 state with rate k on , and back to OFF2 state with rate k off . As this work focuses on the first ON1 phase, subscripts are dropped from hereon.

Article Snippet: For live transcript analysis, we engineered the haploid strains of Saccharomyces cerevisiae (BY4742 and BY4741), which are isogenic to S288C (Research Genetics/Invitrogen, Huntsville, AL).

Techniques: Expressing, Quantitative RT-PCR, Sequencing, Activity Assay, Microscopy, Produced