Transformation of Schizosaccharomyces japonicus
- Microbial Genetics Laboratory, Genetic Strains Research Center, National Institute of Genetics, Mishima, Shizuoka 411-8540 Japan
- ↵1Correspondence: hniki{at}nig.ac.jp
Abstract
This protocol describes the use of electroporation to transform Schizosaccharomyces japonicus with plasmids or linear DNA. Plasmids are helpful for the complementation testing of mutations and for the expression of specific genes. Linear DNA fragments integrated into chromosomal DNA by homologous recombination are useful for gene deletion or to fuse a gene with a tag sequence (e.g., encoding a fluorescent protein). To introduce DNA into S. japonicus, electroporation methods are recommended because S. japonicus is sensitive to lithium acetate (LiOAc).
MATERIALS
Reagents
Agar plates for selection of transformants (see Steps 14–15)
DTT (1 m)
Milli-Q water, sterilized and ice-cold
Plasmid or linear DNA fragment (in distilled water or 1/2× TE buffer; see Step 8)
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Plasmids specific for transformation of S. japonicus (Aoki et al. 2010) were constructed using an autonomously replicating sequence (ARS) isolated from the S. japonicus genome.
Schizosaccharomyces japonicus strain of interest
Sorbitol (1 m)
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YE liquid medium is required. If necessary, the medium can be supplemented with 0.1 mg/mL adenine and 0.1 mg/mL uracil (see Step 13).
Equipment
Electroporation cuvettes (0.2-cm), prechilled to 4°C
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We use those recommended for the Gene Pulser Xcell system.
Electroporation system (e.g., Gene Pulser Xcell [Bio-Rad])
Incubator
Shaking water bath incubator and tubes at 30°C
Tabletop centrifuge
METHOD
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Most electroporation systems have an application guide for S. pombe. The procedures described for S. pombe can generally be used for S. japonicus. The major modification required for S. japonicus is washing with a solution containing 1 m sorbitol and 50 mm DTT to prepare electrocompetent cells. The electroporation pulse parameters should be performed as described in the instruction manual provided by the manufacturer.
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1. Inoculate a strain of interest in 3 mL of YE medium in 18-mm test tubes and shake overnight at 30°C.
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The culture must be saturated.
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2. Subculture an overnight culture by diluting 1:50 or 1:100 in 25 mL of YE medium in a 200-mL flask. Cultivate to early log phase (5 × 106 cells/mL) in YE medium at 30°C with gentle shaking.
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3. Chill the cells on ice for 15 min and harvest (1100g for 3 min at 4°C in a tabletop centrifuge).
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4. Wash cells three times with 10 mL of sterilized, ice-cold Milli-Q water. Centrifuge at 1100g for 3 min at 4°C after each wash.
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Cells are washed to remove salt left from the culture medium.
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5. Resuspend the cells in 10 mL of 1 m sorbitol and add 500 µL of 1 m DTT (for a final DTT concentration of ~50 mm). Incubate the suspension for 15 min at 30°C, and then centrifuge for 3 min at 1100g and 4°C.
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6. Wash the cells once in 5 mL of ice-cold 1 m sorbitol and centrifuge at 1100g for 3 min at 4°C.
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7. Remove as much of the supernatant as possible, and resuspend the cells in 1 mL of ice-cold 1 m sorbitol with gentle pipetting.
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8. Transfer an aliquot of the cell suspension (~1.1 mL is recommended) into a microcentrifuge tube. Centrifuge at 1100 g for 3 min at 4°C to remove the supernatant. Add DNA (up to 0.5 µg in distilled water or 0.5× TE buffer) into the microcentrifuge tube and mix by gently pipetting.
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9. Adjust the cell slurry to 50 µL using sterilized, ice-cold Milli-Q water, and incubate on ice for 30 min.
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10. Transfer 50 µL of the DNA-cell mixture to a prechilled (4°C) 0.2-cm electroporation cuvette.
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11. Place the cuvette into the electroporation chamber. Pulse once under the following conditions: 2.3 kV, 200 Ω, and 25 µF.
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12. Remove the cuvette from the chamber and immediately add 1 mL of ice-cold 1 m sorbitol to the cuvette.
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13. Transfer the cell mixture from the cuvette to 10 mL of YE medium and leave overnight at 30°C.
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When the cells require uracil or adenine, supplementation of YE with 0.1 mg/mL uracil or adenine is recommended.
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14. Plate aliquots of the electroporated cells on appropriate selection agar plates.
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15. Incubate the plates for an appropriate period: 2–3 d for selection by antibiotic resistance, or 1 wk for selection by nutrient requirement.
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False-positive colonies often grow on YE medium when G418 is used for selection.
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Footnotes
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From the Fission Yeast collection, edited by Iain M. Hagan, Antony M. Carr, Agnes Grallert, and Paul Nurse.
