Scarless deletion by intrachromosomal homologous recombination.

<p>(A) Strategy. The target gene is replaced by recombineering using a landing pad amplified using primers containing homology regions A, B, and C. The landing pad is then eliminated by <i>in vivo</i> I-SceI digestion and λ-Red mediated homologous recombination between regions C, followed by counterselection against <i>tetA</i> landing pad retention with 6 mM NiCl₂. (B) Verification of the deletion by colony PCR using primers flanking the <i>rrnB</i> operon. Lane 1: wildtype <i>rrnB</i> from MG1655 (WT; ~6 kbp); Lane 2: MG1655 <i>rrnB</i>::<i>tetA</i> landing pad integrant (LP; ~1.6 kbp). Lane 3–10: 8 randomly picked colonies after deletion (270bp). (C) Sequence of the operon <i>rrnB</i> and the sequencing result after deletion. Targeted homology regions are indicated by same color scheme as in (A).