Figure 1: Two-step allelic exchange. | Nature Protocols

Figure 1: Two-step allelic exchange.

From: Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange

Figure 1

Host cell proteins drive homologous recombination between the allelic exchange vector and the recipient chromosome. The asterisk denotes any insertion, deletion or base substitution, which may be large (>10 kb for deletions) or as small as a single nucleotide. Dashed lines indicate the outcomes of homologous recombination that are selected at each of the two key steps of allelic exchange. After first crossover, which occurs shortly after conjugation, antibiotic selection (Step 36) is used to select for merodiploids. Subsequently, sucrose counter-selection (Step 37) is used to select for double crossovers. Depending on the physical locus of the second crossover, recombination will either restore the wild-type allele or fix the mutant allele in the bacterial chromosome. A third but rare possible outcome is counter-selection escape. This outcome produces a sucrose- and antibiotic-resistant merodiploid, resulting from an inactivating mutation in sacB.

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