Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli

BMC Molecular Biology

Table 3 Generation of EHEC 0157:H7 unmarked deletions of eae and tir

Exp	Substrate Preparation	Amount of DNA	Genotype of linear fragment	Genotype of recipient	Percent Suc^R Cam^Stransformants^a	Recombinants verified by PCR (# verified/# tested)
1.	Digest of pKM182 (SacI, SphI)	2 μg	Δeae-cesT-tir	Δeae-cest-tir::cat-sacB	94% (94/100)	6/6
2.	Digest of pKM181 (SacI, SphI)	2 μg	Δeae	Δeae::cat-sacB	88% (88/100)	6/7
3.	Purified Kpn I fragment from pKC5	0.1 μg	Δtir	Δtir::cat-sacB	10.5% (2/19)	2/2

a) The selection for Suc^R-Cam^S transformants (containing precise in-frame deletions) was performed as described previously for E. coli K-12 [3].

ISSN: 1471-2199