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

BMC Molecular Biology

Table 1 λ Red-promoted PCR-mediated replacement of five targeted EHEC O-islands^a

Targeted Island	Sequence numbers^b	Gene(s) putative functions	# of colonies ^c(half the culture)	Growth on kanamycin ^d(# Kan^R/# streaked)	PCR analysis (# verified/# tested)
O-island #12	353934 – 358187	eaeH putative adhesin	10	8/10	3/3
O-island #77	2710424 – 2712382	Z3025 unknown function	37	10/10	4/6
O-island #103	3315828 – 3317575	Z3664 putative virulence protein	18	18/18	2/3
O-island #130–131	4255687 – 4256420	hopD putative enzyme; degradation	254	18/20	3/3
		Z4695 putative iron storage
		yheA unknown function
O-island #169	5311698 – 5313445	Z5815 putative transposase	5	5/5	2/5
		Z5816 putative virulence protein

a) O157::H7 genetic elements not present in E. coli K-12 b) Genbank accession number AE005174 [30]. c) An estimated 0.25 μg of DNA (3.5 μl) was mixed with 50 μl of electrocompetent EHEC cells and shocked as described in Methods section. Cells were suspended in 3 ml LB following electroporation and grown at 37° for 90 min. Half the culture (1.5 ml) was concentrated by centrifugation and spread on LB plates containing 20 μg/ml kanamycin. In the same experiment, a PCR fragment containing the ΔlacZ::kan allele generated 272 Kan^R transformants. d) Following 24 h growth, candidates were restreaked on LB-kan plates. Among these Kan^R candidates, recombinant deletion formation was verified by PCR analysis as described in Methods section (data not show).

ISSN: 1471-2199