Figure 1

Main plasmid-based methodologies for chromosomal integration of parts in E. coli . A) Homologous recombination. The integrative plasmid (green) carries a sequence (red) that is homologous with a region (red) in the bacterial chromosome (blue). After a single-crossover event, mediated by the endogenous recombination machinery of E. coli, the whole plasmid sequence is integrated in the target region of the chromosome. In specific recA-knockout strains the recombination machinery can be expressed via a helper plasmid. B) Site-specific recombination. The integrative plasmid carries a bacteriophage attachment site (attP) that targets the whole plasmid into the specific attachment site (attB) in the host genome. This process is mediated by a specific recombinase that can be expressed via a helper plasmid. The attP and attB sites are composed by the POP’ and BOB’ sequences, respectively. They share a homologous core sequence (O) and different flanking sequences (P, P’, B and B’), so that after site-specific recombination the integrated plasmid is flanked by the BOP’ and POB’ sequences. All the bacteriophage specific att sequences have this common structure. In both homologous and site-specific recombinations, positive integrants are usually selected via antibiotic resistance, provided that the integrative plasmid sequence contains an antibiotic resistance marker.