Fig. 2

Locus specific PCR to confirm the replacement of grlRA ⁺ with ∆grlRA::kan allele (a) and hfq ⁺ with ∆hfq::cat (b) in E. albertii by recombineering. a – Three colonies were selected from LB plates supplemented with kanamycin at 20 and at 22.5 μg/ml and screened by PCR using the primer pair SB2470 and SB2471 to confirm that the Kan^R phenotype arose from the acquisition of the ∆grlRA::kan cassette and the concomitant loss of the grlRA ⁺ locus. An aliquot (4 μl) of each PCR reaction was electrophoresed and visualized essentially as described previously. All the Kan^R isolates (lanes 2–7) tested positive for the mutant ∆grlRA::kan allele (~2.3 kb) and negative for the wild type allele (~1.8 kb). Lane 1 – 1 kb TrackIt^TM Plus DNA Ladder; Lanes 2–7 – candidate ∆grlRA::kan recombinants isolated on 20 μg/ml (lanes 2–4) or 22.5 μg/ml kanamycin (lanes 5–7). Lane 8 – wild type grlRA ⁺ allele. b – The only colony observed on the Cm plate (6.25 μg/ml) was verified by PCR, using the primer pair SB2485 and SB2486, to confirm the replacement of the hfq ⁺ allele (~527 bp) with the ∆hfq::cat allele (~1.27 kb). Lane 1–1 kb TrackIt^TM Plus DNA Ladder; Lanes 2–3 – replicate PCR reactions with the solitary Cm^R isolate; Lanes 4–5 – replicate PCR reactions for the wild type hfq ⁺ allele