An Integrated System for Precise Genome Modification in Escherichia coli
Fig 5
Scarless deletion by intrachromosomal homologous recombination.
(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 in vivo I-SceI digestion and λ-Red mediated homologous recombination between regions C, followed by counterselection against tetA landing pad retention with 6 mM NiCl2. (B) Verification of the deletion by colony PCR using primers flanking the rrnB operon. Lane 1: wildtype rrnB from MG1655 (WT; ~6 kbp); Lane 2: MG1655 rrnB::tetA landing pad integrant (LP; ~1.6 kbp). Lane 3–10: 8 randomly picked colonies after deletion (270bp). (C) Sequence of the operon rrnB and the sequencing result after deletion. Targeted homology regions are indicated by same color scheme as in (A).