Dual In/Out method for plasmid-less marker-less strain construction. A: deletion of the native φ80-attB by Red recombination. B: Red integration of the φ80-removable marker to the desired loci of MG-Δ(φ80-attB) chromosome. C: curing of the marker by φ80-Int/Xis-system. D: φ80-driven integration of the CRIM plasmid with target cassette into the different of φ80-attB sites. E: construction of "marker-less" cassette-carrier-strains by λ-Int/Xis excision of the vector part of integrative plasmids.