Abstract
An rps12-mediated gene replacement method in a cyanobacterium Synechococcus elongatus PCC 7942 was developed previously using a wild-type rps12 gene encoding S12 ribosomal protein that acts as a dominant streptomycin-sensitive marker. An improved version of this method is described here utilizing a heterologous rps12 gene from a cyanobacterium Synechocystis sp. PCC 6803.
Heterologous rps12 gene was inserted at psbAI gene as a target in a host strain of PCC 7942 resistant to streptomycin, resulting in streptomycin-sensitive merodiploids. Gene replacement in the merodiploids allowed selection of 50% recombinants. As streptomycin-sensitive phenotype of the merodiploids was stabilized under the strong promoter upstream of the heterologous rps12 gene, a cassette containing the psbAI promoter-rps12 gene fusion with a kanamycin resistance gene was constructed, which was capable of insertion to/removal from chromosome and suitable for gene replacement free from markers.
