Figure 1

Basic principle of introducing mutations into the bacterial chromosome using rpsL -based counter-selection. Prerequisite for the used strain is a chromosomal resistance against streptomycin conferred by a mutation in rpsL. If necessary, the strain can be made Strep^R before by homologous recombination of a mutated rpsL gene (e.g. rpsL150) (step 1). Around the point of interest within the target gene, the rpsL-neo cassette is inserted via 50 bp homology arms by Red^®/ET^® Recombination (step 2). Positive clones are Kan^R. Due to the additional wild-type allele of rpsL, the strain becomes Strep^S (step 3). In the next step, the rpsL-neo cassette is replaced by Red^®/ET^® Recombination against the modified double-stranded (ds) or single-stranded (ss) DNA-fragment of the target gene (carrying the point mutation) (step 4). Positive clones become Strep^R again and can therefore easily be selected (step 5). The asterisk represents the point mutation within the target gene.