Table 2.
Summary of the thyA-null experiment, rescue assays, first and second stages of Col10a1 mutagenesis
| Experiment | Bacterial strain and DNA fragment/plasmid used | Induction of λ-Red system | Total number of colonies | No. of correctly targeted colonies relative to the number of colonies screened | Selection efficiency (%) |
|---|---|---|---|---|---|
| thyA-null strain (QW1) | DY380 + ΔthyA (PCR) | + | 255 | 11 out of 12 | 92 |
| DY380 + ΔthyA (PCR) | − | 2 | 0 out of 2 | — | |
| Rescue assays | QW1 + pThyA-FL | − | 1346 | — | — |
| QW1 + pThyAΔ3′ | − | 1386 | — | — | |
| QW1 + thyA-FL (PCR) | − | 0 | — | — | |
| QW1 + thyA-Δ3′ (PCR) | − | 0 | — | — | |
| First stage of Col10a1 mutagenesis | QW1-pKM2-ColX + X-thyAΔ3′-X′ (PCR) | + | 2500 | 12 out of 13 | 96 |
| − | 1 | 0 out of 1 | — | ||
| Second stage of Col10a1 mutagenesis | QW1-(X-thyAΔ3′-X′) + ColX-G10D (PCR) | + | 1260 | 14 out of 16 | 94 |
| − | 1 | 0 out of 1 | — |
The values given are the result from a typical experiment, while the selection efficiency percentage is the mean of three independent experiments. PCR denotes DNA fragments generated by PCR.