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ProtocolsYSD Pipeline
Yeast Transformation & Gap Repair
Standard operating procedure from the WeaveSeq experiments team, shared across the YSD pipeline runs.
Standard operating procedure from the WeaveSeq experiments team, shared across the YSD pipeline runs.
Co-transform linearized pCTCON2 vector and dsDNA oligo pool into EBY100 yeast via electroporation. The oligos repair the gapped vector by homologous recombination, building the display library in vivo.
This protocol follows Benatuil et al. (2010) Protein Eng Des Sel 23:155–159, which achieves 1–1.5 × 10⁸ transformants/µg vector DNA and can generate libraries of 10⁹–10¹⁰ in a single day. It is the gold standard for YSD library construction.
| Buffer | Composition | Notes |
|---|---|---|
| YPD | 20 g/L peptone, 20 g/L dextrose, 10 g/L yeast extract | Rich medium |
| Electroporation buffer | 1 M sorbitol, 1 mM CaCl₂ | Ice-cold, sterile |
| Conditioning buffer | 0.1 M LiAc, 10 mM DTT | Pre-warmed to 30°C; prepare fresh |
| Recovery medium | 1:1 (v/v) 1 M sorbitol : YPD | Pre-warmed to 30°C |
| SD-CAA plates | See SOP 05 for recipe | Selective agar |
See SOP 01 for full details.
See SOP 03 for full details.
DNA ratio: Use 1:3 vector:insert molar ratio (4 µg vector + 12 µg insert per cuvette is standard for a 6 kb vector with 300 bp inserts).
Dilute overnight culture into 100 mL fresh YPD to OD₆₀₀ = 0.3.
Grow at 30°C, 250 rpm until OD₆₀₀ = 1.3–1.6 (early-to-mid log phase, ~3–5 hours).
Harvest cells: centrifuge 3,000 × g, 5 min, 4°C.
Wash 2× with 50 mL ice-cold sterile water. Resuspend completely each time.
Wash 1× with 50 mL ice-cold electroporation buffer (1 M sorbitol/1 mM CaCl₂).
Resuspend in 20 mL conditioning buffer (0.1 M LiAc/10 mM DTT, 30°C).
Incubate at 30°C, 30 min, 250 rpm. ⚠️ This step is critical. Omitting LiAc/DTT causes 86–93% loss of transformation efficiency.
Pellet cells. Wash 1× with 50 mL ice-cold electroporation buffer.
Resuspend in electroporation buffer to ~1.6 × 10⁹ cells/mL (typically 100–200 µL total from a 100 mL culture).
In a pre-chilled 1.5 mL tube, combine:
Transfer to pre-chilled 0.2 cm gap cuvette. Keep on ice.
Electroporate at: 2.5 kV (12.5 kV/cm), 25 µF capacitance.
Immediately after pulse: add 1 mL pre-warmed recovery medium (1:1 sorbitol:YPD) to cuvette. Resuspend gently.
Transfer to culture tube. Add recovery medium to ~10 mL total.
Incubate at 30°C, 1 hour, 250 rpm.
Pellet cells, wash 1× with SD-CAA.
For titer: Plate serial dilutions (10⁻⁴, 10⁻⁵, 10⁻⁶) on SD-CAA plates. Incubate 30°C, 2–3 days. Count colonies to determine transformation efficiency.
For library: Inoculate remaining cells into liquid SD-CAA at OD₆₀₀ ~0.2–0.3 for expansion.
Critical segregation step: After electroporation, ~85% of colonies initially contain multiple plasmids per cell. Passage the pooled library in liquid SD-CAA for ≥2 doubling times before plating or sorting to allow plasmid segregation.
| Library Diversity | Target Coverage | Transformants Needed | Cuvettes Needed* |
|---|---|---|---|
| 10³ (384 oligos) | 100× | 3.8 × 10⁴ | 1 |
| 10⁴ | 100× | 1 × 10⁶ | 1 |
| 10⁵ | 100× | 1 × 10⁷ | 1 |
| 10⁶ | 10× | 1 × 10⁷ | 1 |
| 10⁷ | 10× | 1 × 10⁸ | 1–2 |
| 10⁸ | 10× | 1 × 10⁹ | 10–20 |
* Assuming 1 × 10⁸ transformants per cuvette at Benatuil efficiency.
Always maintain ≥10× oversampling of the estimated library diversity at every passage step. Never passage fewer than 10× the estimated unique clone count.
| Check | Pass Criterion |
|---|---|
| Transformation efficiency | ≥5 × 10⁷ CFU/µg vector |
| Vector-only control | 0 colonies |
| Library diversity coverage | ≥10× (confirmed by colony count) |
| Cell viability post-electroporation | ≥50% by trypan blue (optional) |
| Problem | Likely Cause | Solution |
|---|---|---|
| Low efficiency (<10⁷/µg) | Incomplete LiAc/DTT step | Verify conditioning buffer is fresh; confirm 30 min incubation |
| Cells overgrown (OD >2.0) | Harvest at OD 1.3–1.6 | |
| Salt in DNA prep | Ethanol-precipitate and wash DNA 2× with 70% ethanol | |
| Arcing | Conductive contamination | Re-purify DNA; ensure cuvettes are dry externally |
| High vector-only background | Incomplete digest | Run digest to completion; gel-purify; triple-digest (NheI + BamHI + stuffer cutter) |
| Polyclonal colonies | Insufficient segregation time | Passage library ≥2 doublings before analysis |