Table 3 Summary of protocol for high-level electrotransformation of C. pasteurianum and comparison to initial low-level protocol

Cell growth

 Growth additive

None

1.25% glycine (at OD₆₀₀ 0.3-0.4)

 Osmotic stabilizer

None

Hypertonic sucrose (0.4 M; at OD₆₀₀ 0.3-0.4)

 OD₆₀₀ at time of harvest

OD₆₀₀ 0.6-0.8

OD₆₀₀ 0.6-0.8

Washing and pulse delivery

 Osmotic stabilizer

Isotonic sucrose (0.27 M)

Isotonic sucrose (0.27 M)

 Cell membrane solubilizer

None

5% (v/v) ethanol 5 min prior to pulse

 DNA amount

5 μg

0.5 μg

 Pulse parameters

2.0-2.5 kV; 25 μF; 200–800 Ω; 6–9 ms

1.8 kV; 25 μF; ∞ Ω; 12–14 ms

Outgrowth

 Osmotic stabilizer

None

Hypotonic sucrose (0.2 M)

 Recovery time

16 h

4-6 h

 Transformation efficiency

2.4 × 10¹ transformants μg^-1 pMTL85141

Up to 7.5 × 10⁴ transformants μg^-1 pMTL85141