2.1 Competent Yeast Cells Are Sensitive to Osmotic Stress After Heat-Shock

In the original protocol by Schiestl and Gietz (1989), yeast cells are centrifuged after incubation at 42°C, washed in TE buffer, and plated on agarose plates. Their optimized method, which achieves yields of up to 10⁶ transformants per µg of plasmid DNA (Gietz et al. 1992), includes the same final steps. Subsequent iterations of this efficient protocol proposed by Gietz and Schiestl (2007a, 2007b) also incorporate the washing step, although sterile water is used instead of TE for plating the yeast cells.

In all these protocols, yeast cells are washed in a hypotonic solution. To assess the resilience of the permeabilized yeast cells under these conditions, we examined their viability in water versus 2 M sorbitol. In one experiment, six aliquots (50 µL each) of BY4742∆TRP1 yeast cells, resuspended in LiAc–TE, were incubated for 30 min at 30°C in the presence of ssDNA and PEG, followed by a heat shock for 15 min at 42°C. After centrifugation and removal of the PEG/LiAc–TE/ssDNA mixture, the pelleted cells were resuspended in either 1 mL of sterile water or 1 mL of 2 M sorbitol. Following serial dilution (in water or sorbitol), two aliquots (100 µL each) of cells were plated on YPD medium. The number of colonies was counted after 3 days of incubation at 30°C. This experiment was repeated twice with two independent yeast cultures, and Table 1 presents the average values. The results (Figure 1) indicate a significant loss of viability when treated yeast cells are resuspended in water, as evidenced by the ~50% reduction in colony formation compared to yeast cells resuspended in 2 M sorbitol (Wilcoxon rank sum test, W = 31, p value = 0.048).

2.2 Yeast Cells Are Sensitive to Osmotic Stress When Acquiring Their Competence

Next, we investigated the osmotic sensitivity of yeast cells during incubation in the presence of PEG. Two solutions of 50% PEG were utilized: the first (S) corresponded to the standard protocol (50% PEG in water), while the second (S*) was prepared in sorbitol, achieving a final concentration of 1.2 M. Additionally, we supplemented the standard mix (50% PEG in water) with either 90 µL of water (S + H) or 2 M sorbitol (S + S).

Yeast cells were incubated in these various mixtures for 30 min at 30°C in the presence of ssDNA and plasmid DNA (80 ng) before undergoing a heat shock for 15 min at 42°C. In all cases, the transformed yeast cells were resuspended in 2 M sorbitol. We assessed both cellular viability (by plating on complete medium) and transformation efficiency (by plating onto selective medium).

Table 2 presents the values plotted in Figure 2. Notably, the addition of water (i.e., reducing osmotic pressure) resulted in decreased cellular viability (S vs. S + H, panel A). Conversely, the addition of sorbitol enhanced viability (S + S vs. S, panel A). This trend was also observed, albeit to a lesser extent, with the modified mix (50% PEG, 1.2 M sorbitol) (S* vs. S, panel A).

Transformation efficiency exhibited a similar pattern, except for one condition: S* (panel B). Surprisingly, at high concentrations of sorbitol and PEG, we were unable to get any transformants. This complete inhibition of transformation is reproducible. However, the addition of sorbitol to the standard mix significantly improved transformation efficiency (S + S vs. S, panel B).

2.3 Optimization of Sorbitol Concentration

Since sorbitol enhances cell viability, we subsequently measured this effect at various concentrations. In a first set of experiments, we compared the sensitivity of competent cells to solutions ranging from 0 to 2 M sorbitol. Six aliquots (50 µL each) of BY4742∆TRP1 yeast cells, resuspended in LiAc–TE, were incubated for 30 min at 30°C in the presence of ssDNA, LiAc–TE, and PEG, followed by a heat shock for 15 min at 42°C. After centrifugation and removal of the PEG/LiAc–TE/ssDNA mix, the pelleted cells were resuspended in either 1 mL of sterile water or sorbitol at different concentrations (0.5, 1, 1.5, and 2 M). Following serial dilution (in water or sorbitol), three or four aliquots (50 µL each) of cells were plated on YPD medium. The number of colonies was counted after 3 days of incubation at 30°C. The results presented in Table 3 derive from three independent assays. The corresponding box-plot (Figure 3A) illustrates the protective role of sorbitol, with the most effective concentration being 0.5 M.

Next, we optimized the concentration of the sorbitol added in the transformation mix. Competent yeast cells (50 µL) were incubated with the standard mix (356 µL PEG 40%, LiAc– TE, 25 µg ssDNA, 80 ng plasmid) + 90 µL of H₂O or sorbitol at different concentrations. After heat-shock, yeast cells were resuspended in 1 mL of 0.5 M sorbitol. The transformation efficiency was determined by plating several aliquots of 50 µL on selective medium. The results presented in Table 4 derive from two independent experiments with two different cultures. The corresponding box-plot (Figure 3B) demonstrates a protective role for sorbitol. This protective effect remains roughly the same at concentration ranging from 1 to 2 M.

We combined our two observations—protection by sorbitol during incubation and during cell recovery—and compared this optimized protocol with the standard protocol.

2.4 Two Slight Modifications Improve Yeast Transformation Rate

The modified protocol was assessed with two yeast strains: W303-1b and BY4742ΔTRP1. For each assay, 50 mL of cell culture (OD_650nm≈1) was collected, washed, and resuspended in 250 µL of a buffer containing lithium acetate (LiAc, 0.1 M), Tris-HCl (50 mM, pH 7.4), and EDTA (1 mM). In the standard condition, 50 µL of competent cells were mixed with 350 µL of a solution, consisting of 280 µL of 50% polyethylene glycol (PEG) and 70 µL of 5X LiAc–TE, along with 5 µL of DNA carrier (25 µg) and 1 µL of plasmid DNA (80 ng). After heat shock, the yeast cells were suspended either in 1 mL of water (H) or in 1 mL of 0.5 M sorbitol (S).

In parallel, 50 µL of competent cells were mixed with 350 µL of the standard solution in which 90 µL of 2 M sorbitol was added. After heat shock, the yeast cells were suspended in 1 mL of 0.5 M sorbitol (SS).

A volume of 100 µL from each suspension was then spread onto selective medium, with several plates prepared for each experimental condition. The modified protocol incorporating sorbitol (SS) demonstrated a significant enhancement in transformation efficiency compared to the standard protocol (H) across a dozen independent experiments. This improvement was consistent regardless of the yeast strain used (data not shown). However, the level of improvement was highly variable ranging from 2 to 10. This lack of reproducibility coincides each time with the use of new PEG or sorbitol solutions. We checked the impact of the water, the sterilization process (filtration vs. sterilization) and the origin of the chemical products, but were unable to find any explanation for this discrepancy. In all cases, the greatest effect observed was due to the recovery of cells in a 0.5 sorbitol solution (S vs. H vs. SS). We therefore decided to analyze the results using the batch of compounds that led to the least favorable outcome. We also adjusted the volume of 2 M sorbitol added to the standard mix (40 µL instead of 90 µL) to maintain a protective effect while retaining a high PEG concentration. Table 5 shows the results obtained with three independent cultures of BY4742ΔTRP1 and three independent cultures of W303-1b.

The corresponding box-plots (Figure 4) highlight the capacity for the sorbitol solution to increase transformation efficiency. Interestingly, the genetic background has a strong impact on the results. With strain BY4742ΔTRP1, the sorbitol added in the transformation mix has no significant effect (panel A SS vs. S) while the use of sorbitol for cell suspension increases by a factor of around two the transformation efficiency (H vs. S or SS). In contrast, W303-1b is much more sensitive to sorbitol (panel B) and the optimized protocol increases transformation efficiency by a factor of around 8 (SS vs. H). Interestingly, adding sorbitol in the transformation mix seems to decrease the variability of the results (SS vs. S).

4.1 Yeast Strains, Plasmid, and Growth Media

The two yeast strains used are W303-1b {MATα leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15} and BY4742∆TRP1 {MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 YDR007W::kanMX}, obtained from the commercial EUROSCARF knockout library.

Transformations were performed using pAG424-ccdB (Addgene), in which the ORF of the URE2 gene replaces the ccdb cassette, as described by Alberti et al. (2007).

Yeast strains were routinely grown in YPD (1% yeast extract, 2% peptone, and 2% glucose in distilled water), and transformed yeast was selected on SC-TRP (CSM-Trp (Formedium) 740 mg/L, YNB (DIFCO) 6.7 g/L, Glucose 2%, Agar 2%). With the exception of sorbitol (Formedium), all chemical products were sourced from Sigma.

4.2 Transformation

In total, 50 mL of YPD were inoculated with 1 mL of an overnight saturated culture (in YPD) and incubated at 30°C until the culture reached an OD_650nm of 1. The culture was centrifuged (5′ at 3000 rpm) and washed with 50 mL of sterile water. After centrifugation (5′, 3000 rpm), yeast cells were resuspended in 1 mL of LiAc–TE solution (0.1 M lithium acetate, 50 mM Tris-Cl pH 7.4, and 1 mM EDTA) and transferred to an Eppendorf tube. The suspension was centrifuged (5′, 3000 rpm), and the yeast pellet was resuspended in 250 µL of LiAc–TE solution.

In total, 50 µL of the resuspended cells were mixed with 280 µL of PEG 3500 (50% W/V), 70 µL of 5X LiAc–TE (0.5 M LiAc, 250 mM Tris-Cl pH 7.4, 5 mM EDTA), and 25 µg of herring genomic ssDNA and 80 ng of plasmid.

In the modified protocol, 40 µL of 2 M sorbitol solution were added to this mix.

The tube was then incubated at 30°C for 30 min and heat-shocked at 42°C for 15 min. After centrifugation (5′, 3000 rpm), yeast cells were resuspended either in 1 mL of water (standard protocol) or in 1 mL of 0.5 M sorbitol (modified protocol).