Integrative modules for efficient genome engineering in yeast

Abstract

We present a set of vectors containing integrative modules for efficient genome integration into the commonly used selection marker loci of the yeast Saccharomyces cerevisiae. A fragment for genome integration is generated via PCR with a unique set of short primers and integrated into HIS3, URA3, ADE2, and TRP1 loci. The desired level of expression can be achieved by using constitutive (TEF1p, GPD1p), inducible (CUP1p, GAL1/10p), and daughter-specific (DSE4p) promoters available in the modules. The reduced size of the integrative module compared to conventional integrative plasmids allows efficient integration of multiple fragments. We demonstrate the efficiency of this tool by simultaneously tagging markers of the nucleus, vacuole, actin, and peroxisomes with genomically integrated fluorophores. Improved integration of our new pDK plasmid series allows stable introduction of several genes and can be used for multi-color imaging. New bidirectional promoters (TEF1p-GPD1p, TEF1p-CUP1p, and TEF1p-DSE4p) allow tractable metabolic engineering.

Keywords: Saccharomyces cerevisiae; bidirectional promoter; genetic integration; integrative plasmid; vector; yeast.

Figure 1. FIGURE 1: pDK vector series overview.

(A) pDK vector with an integrative module flanked by a split marker. The customized insert is flanked by constitutive, inducible, or daughter-specific promoters. PCR is required for genetic integration. (B) Integration overview: the module is transformed into S. cerevisiae and inserted into the chromosome resulting in a marker locus duplication. (C) pDK vector set: constitutive, inducible, and four bi-directional promoter plasmids available for integration into four markers.

Figure 2. FIGURE 2: Proof of concept - Organelle inheritance order during division.

(A) In vivo 4-color imaging of peroxisome (SKL signal C-terminally fused to 4 mCherry (mCH) on pDK-UT), actin (LifeAct fused to GFP on pDK-AT), nucleus (SV40 nls signal fused to 3 far-red fluorophores on pDK-HT), and vacuole (VPH1 endogenously tagged with mBFP), the scale bar is 1 µm. The graph represents proximity of peroxisome to actin cable during division, Fluorescence Intensity (FI) was calculated along the x axes (arrow on the merge image) for red (peroxisome) and green (actin) channels. (B) The timeline of organelle inheritance in the wild type (wt) strain, scale bar is 1 µm, time points represent the average (n = 30) time of organelle inheritance from the start of division, strain carries SKL signal C-terminally fused to 4 mCherry (mCH) on pDK-UT module, SV40 nls signal fused to 3 far-red fluorophores on pDK-HT module, and VPH1 endogenously tagged with GFP. (C) The timeline of the organelle inheritance in the Δinp2 strain, scale bar is 1 µm.

Figure 3. FIGURE 3: Proof of concept - Concentration does not correlate with inclusion formation.

(A) Gradual increase in the amount of GFP-VHL according to integrated copies, Fluorescence Intensity was quantified in 30 single cells in the population, the average and standard errors are represented on the graph. (B) Confocal images of cells carrying 4 copies of GFP-VHL on pDK-AC, TC, UC, HC modules subjected to the range of temperatures for 1h, scale bar 1 µm. (C) Quantification of inclusion forming cells in the population according to the change in the temperature and the concentration, the numbers represent % of the cells with inclusions (n = 300), correlation coefficient of aggregation and temperature (r(temperature)) and aggregation and concentration (r(concentration)) is provided under the diagram.

Figure 4. FIGURE 4: Bidirectional promoter reporters.

(A) Galactose inducible bidirectional promoter, cells carrying pDK-HGG-GFP-mCherry were induced with galactose or grown on glucose for 6 hours, scale bar 1 µm. (B) Constitutive-inducible promoter, cells carrying pDK-HTC-GFP-mCherry were grown with and without (control) copper2+ for 4 hours, scale bar 1 µm. (C) Constitutive bidirectional promoter, cells carrying pDK-HTG-GFP-mCherry were grown on glucose containing medium to middle log phase, scale bar 1 µm. (D) Daughter-specific-constitutive bidirectional promoter, cells carrying pDK-HTD-GFP-mCherry module were grown on glucose containing medium on the microscope, frames are shown (0 min, 60 min, 100 min). (E) Comparison of fluorescence intensity of bidirectional reporters, the graph shows the average of fluorescence intensity of 20 cells and standard error.