Abstract
Markerless chromosomal in-frame deletion mutants are often used to avoid polar effects, conserve antibiotic resistance markers or make multiple mutations. This protocol capitalizes on the sacB gene (lethal in the presence of sucrose), for selection against a single cross-over event, on a suicide vector (contains no Rhodobacter capsulatus compatible origin of replication). The bacterial strains, media and suicide vectors in this protocol are specific for R. capsulatus, but the concepts can be applied to other species.
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