TABLE 4.
Optimization of electrotransformation for B. smithii ET 138
| Parameter change | Final ODa | Period of growth (h)b | Electroporation setting |
Cuvette size (mm) | Amt (μg) of DNA | Recovery mediumc | CFUd | ||
|---|---|---|---|---|---|---|---|---|---|
| Voltage (kV) | μF | Ω | |||||||
| Settingse | 0.483 | 1.75 | 1.5 | 25 | 600 | 1 | 1 | RG2 | 3 |
| Settingsf | 0.483 | 1.75 | 2.0 | 25 | 200 | 2 | 1 | RG2 | 33 |
| Settings | 0.424 | 2.9 | 2.0 | 25 | 400 | 2 | 0.7 | RG2 | 157 |
| Recovery medium | 0.519 | 1.75 | 2.0 | 25 | 400 | 2 | 1 | LB2 | 960 |
| Recovery medium | 0.519 | 1.75 | 2.0 | 25 | 400 | 2 | 1 | RG2 | 5 |
| Fast growthg | 0.539 | 1 | 2.0 | 25 | 400 | 2 | 2.5 | LB2 | 1,900 |
| μg DNA addedg | 0.617 | 1.3 | 2.0 | 25 | 400 | 2 | 0.2 | LB2 | 2,409 |
| μg DNA addedg | 0.617 | 1.3 | 2.0 | 25 | 400 | 2 | 0.02 | LB2 | 5,118 |
a
Final OD600 after the indicated number of hours when growing cells prior to making them competent.
b
Number of hours cells had grown before making them competent.
c
RG2 is LB2 with 121 g/liter sucrose and 10 g/liter glucose (29).
d
CFU indicate CFU per μg DNA. Using fresh cells or cells stored at −80°C did not change CFU counts.
e
Settings from reference 29.
f
Settings are based on reference 30, the same settings as those used for screening of Geobacillus strains.
g
In these experiments, after overnight growth, cells were transferred into 500-ml Erlenmeyer flasks or 1-liter bottles (having a similar bottom surface) instead of into a 250-ml Erlenmeyer flask to allow for more aeration.