Figure 1

Strategy to insert the araC-Cre cassette into the chromosomal lacZ gene in recA13 strain HB101. (A) Compatible plasmids pBAD75Cre and pGETrec were introduced into E. coli HB101. The plasmid pBAD75Cre contains araC-Cre arabinose-inducible expression cassette flanked by targeting homologies to the lacZ gene. The plasmid pGETrec contains araC-recET-Redγ arabinose-inducible expression cassette comprising a truncated recE gene, the recT gene and the bacteriophage λ Redγ gene. (B) Expression of the recET genes was induced by L-arabinose to enable homologous recombination. Clones with integrations of the pBAD75Cre plasmid into the chromosomal lacZ gene were selected as white LacZ^- colonies on LB agar supplemented with Ap, Cm, X-gal and IPTG at 44°C. (C) Ten LacZ^- colonies were pooled and used for 4 overnight passages in L-arabinose containing medium (dilution 1:5000 at each passage step). Induction of recET expression by L-arabinose enabled excision of the entire pBAD75Cre or its deletion derivative pBAD75Δ through homologous recombination. Excision of pBAD75Δ resulted in the desired bacterial strain HB101Cre.