Table 1.
Transformation mix.
Table 2.
VPS38 deletion and check primers.
Table 3.
Strains and plasmids used in this study.
Fig 1.
Plasmid and cell concentration affect transformation efficiency.
(A) BY4741 cells at OD600 0.5 were transformed with the indicated amount of pRB1 plasmid using our automated methodology with 4-hour heat shock. After transformation, cells were spotted on a –Uracil agar plate. (B) Different OD600 cells were transformed with 100ng pRB1 plasmid and heat shocked at 42°C for 4 hours. (C) Microscopy of BY4741 cells originally transformed either at 0.4 or 1.5 OD600, and examined at an OD of 3.5. Numbers indicate average stress granule (Pab1-GFP, green text) or P-body (Edc3-mCh, red text) foci per cell, and the percentage of Edc3-mCh foci co-localized with Pab1-GFP (white text) (D). Quantification of stress granule (SG) and P-Body (PB) size in (C). Data is presented as mean ± standard deviation of 3 independent experiments; n.s., not significant.
Fig 2.
Transformation efficiency is increased with extended 42°C heat shock periods.
(A) BY4741 cells at OD600 0.5 were transformed with the indicated amount of pRB1 plasmid and incubated at 42°C for 30 minutes (min), 1, 2, 4, and 6 hours (h). Transformants were selected on –Uracil plates. (B) Transformed cells generated from transformation reactions with different 42°C incubation times (1, 2, 4 and 6 hours). Numbers indicate average stress granule (Pab1-GFP, green text) or P-body (Edc3-mCh, red text) foci per cell, and the percentage of Edc3-mCh foci co-localized with Pab1-GFP (white text). (C) Quantification of stress granule (SG) and P-body (PB) size in (B). Data is presented as mean ± standard deviation of 3 independent experiments; n.s., not significant. (D) BY4741 cells transformed with pRB1 plasmid and incubated at 42°C for different time were assessed for elevated mutation rates by development of canavanine resistance. Transformed cells were plated on canavanine media (60 mg/L). Simultaneously, 1/200 diluted amount of cells were coated on the YPD plate. Colony number was counted after 2 days incubation at 30°C. Mutation rates were normalized to the 0-hour heat shock. Data is presented as mean ± standard deviation of 3 independent experiments; n.s., not significant.
Fig 3.
Successful application of high-throughput transformation method to gene deletion.
(A) Generation of VPS38 knockout strain by insertion via homologous recombination of a LEU2 selective marker at the VPS38 locus. (B) Different volumes of 150ng/ul of the LEU2 cassette were transformed into yeast cells. Transformants were plated on—Leucine media. (C) VPS38 deletion was verified using check primers (Table 2) from four single colonies (wild type, 1#: 30 μl spot, 2#: 60 μ spotl, 3#: 120 μl spot). Expected PCR product size of wild type: 1701 bp, VPS38 knockout: 2810 bp. (D) Plate 1 from the non-essential yeast knockout library was cultured and transformed with 4500ng VPS38 knockout LEU2 cassette using our methodology. Transformed cells were cultured in –Leucine SD media for 3 days and spotted on –Leucine media using prongs.
Fig 4.
Schematic model of yeast transformation using liquid handling robot.
Step 1, Inoculate overnight culture to the deep well plate. Grow another 2 ½ -4 hours to recover cells to the mid-log phase. Step 2, Prepare plasmid and transformation mix to transformation. Normally, an OD600 range of 0.4–1.5, ≥100ng plasmid or 4500 ng PCR product is optimal for our automated transformation method. Step 3, Heat shock of the transformants for 3–6 hours. Step 4, Transfer the transformed cells to a liquid selective media plate to grow another 2–4 days. Pin the transformed strains onto appropriate selective media to generate the new library.
Table 4.
Comparison of the two plasmid transformation methods.
Table 5.
Comparison of SGA and high-throughput gene deletion methods.