Table 2 Gene replacements in the tir-cesT-eae region of enterohemorrhagic E. coli 0157:H7 using long homology-containing substrates
From: Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli
Exp # | Substrate preparation | Amount of DNA | Genotype of linear fragment | Total Transformants | Percent transformants Amp sensitive | Recombinants verified by PCR (# verified/# tested) |
|---|---|---|---|---|---|---|
Plasmid digests: | ||||||
1. | restriction digest pKM184 (SacI, SphI) | 2 μg | Δeae::cat | >500 | 11% (4/35) | 1/1 |
2. | restriction digest pKM185 (SacI, SphI) | 2 μg | Δeae-cesT-tir::cat | 103 | 2.6% (2/76) | 1/1 |
3. | restriction digest pKM 187 (SacI, SphI) | 1 μg | Δeae::cat sacB | > 500 | 2.8% (1/35) | 1/1 |
Plasmid digests including backbone: | ||||||
4. | restriction digest pKM184 (SacI, SphI, ApaLI) | 0.5 μg | Δeae::cat | 52 | 57% (4/7) | NT |
5. | restriction digest pKM184 (SacI, SphI, ApaLI) | 10 μg | Δeae::cat | > 500 | 95% (143/151) | 10/10 |
6. | restriction digest pKM185 (SacI, SphI, ApaLI) | 2 μg | Δeae-cesT-tir::cat | 30 | 56% (9/16) | 9/10 |
7. | restriction digest pKM185 (SacI, SphI, ApaLI) | 2 μg | Δeae-cesT-tir::cat | 36 | 50% (9/18) | 7/9 |
Gel-purified fragments: | ||||||
8. | purified Sac I-Sph I fragment from pKM185 | 0.5 μg | Δeae-cesT-tir::cat | 225 | 100% (20/20) | 1/1 |
9. | purified Kpn I fragment from pKC2 | 0.1 μg | Δtir::cat | 6 | NT | 6/6 |
10. | purified Sac I-Sph I fragment from pKM188 | 0.6 μg | Δeae cesT tir::cat sacB | 273 | 100% (40/40) | 1/1 |
11. | purified Kpn I fragment from pKC3 | 0.05 μg | Δtir::cat sacB | 2 | NT | 2/2 |