Fig. 1

a Locus specific PCR to confirm the replacement of ler ⁺ with ∆ler::cat allele in E. albertii by recombineering. Twelve colonies that arose on chloramphenicol plates were screened by PCR, using a pair of primers, one of which bound upstream (SB2456) and the other downstream (SB2457) relative to the recombination site, to confirm that the Cm^R phenotype resulted from the acquisition of the ∆ler::cat allele and the concomitant loss of the ler ⁺ allele. An aliquot (4 μl) of each PCR product was electrophoresed on a 1 % agarose gel and stained with ethidium bromide prior to visualization using a transilluminator (BioRad). All (lanes 2–12), but one (lane 13), isolates had successfully replaced the wild type ler ⁺ allele (~1.8 kb) with the mutant ∆ler::cat allele (~2.5 kb). Lane 1 – 1 kb TrackIt^TM Plus DNA Ladder; lanes 2–13 – candidate ∆ler::cat recombinants; lane 14 – wild type ler ⁺ allele. b – Eviction of the cat cassette from the ∆ler::cat locus. Recombinants were transformed with pFT-K, a thermolabile plasmid that expresses the FLP recombinase enzyme under the control of the TetR repressor. Varying concentration of chlorotetracycline (20–200 μg/ml) was added to derepress the flp gene and the frequency of excisants was calculated. The excision frequency was highest at the lowest chlorotetracycline concentration and gradually decreased with increasing chlorotetracycline concentration. Results depict the mean ± standard deviation from a representative experiment involving two independently isolated recombinants. At least 36 candidate excisants per ∆ler::cat recombinant, or all if fewer arose, were phenotyped to determine the excision frequency. c – Locus specific PCR to confirm the eviction of the cat cassette from the ∆ler::cat recombinants. Excisants that were phenotypically Cm^S were screened by PCR using SB2456 & SB2457 to confirm that resensitization to chloramphenicol occurred due to the loss of the cat cassette. All isolates that were phenotypically Cm^S (lanes 2–7) had evicted the cat cassette and gave a PCR product of the expected size (~1.55 kb) compared to the wild type allele (lane 8; ~1.8 kb). Lane 1 – 1 kb TrackIt^TM Plus DNA Ladder; lanes 2–7 – candidate ∆ler::FRT (Cm^S) excisants; lane 8 – wild type ler ⁺ allele