Figure 1

Schematic representation of the construction of specific deletion mutants in E. coli UTI89. In the first step, homology regions A and B, flanking the deletion, were amplified by PCR. Primers were designed in such a way that fragments were generated with an attB-site on one side and with a restriction enzyme site and an overlap region of 25-30 bp on the other side. Overlap-PCR was subsequently carried out with the attB-containing primers, to join regions A and B. The resulting PCR fragment was used in a BP-reaction with the pDONR221 plasmid. This gave rise to a plasmid containing the overlap-PCR fragment. After PmeI restriction of the plasmid, the chloramphenicol resistance marker cat was inserted. A final PCR-fragment was obtained using primers SeqLA1 and SeqLB, which are located on pDONR221. This fragment was electroporated in arabinose-induced E. coli strains harbouring the helper plasmid pKD46 expressing the Red recombinase.