Figure 2

Assembly and integration of the hygromycin B resistance cassette into the genome of X. dendrorhous by DNA assembler. A) Three DNA fragments, DHS3 “up”, hph expression cassette (composed by EF-1α promoter, hph gene and gpd terminator) and DHS3 “down” were amplified by PCR to direct the integration of the hygromycin B cassette into the DHS3 locus of the X. dendrorhous genome. Then, they were co-transformed, assembled (by recombination at their overlapping ends) and integrated into the X. dendrorhous genome. The arrows represent the primers used and the black crosses represent the in vivo homologous recombination event. By this way, the heterozygous strain was obtained, which was submitted to the double recombinant method (DRM) to obtain the homozygous strain. B) Evaluation of the hygromycin B cassette integration by PCR of strains Xd_1H (1H, one hygromycin B cassette copy), Xd_2H (2H, two hygromycin B cassette copies) and parental UCD 67–385 (WT), and control without DNA (-). A scheme representing the primer sets (in arrows and numbers according to Additional file 1: Table S1) that were used and the DNA target are included under each gel photograph. M: Molecular marker, lambda DNA digested with HindIII. C) Color phenotype of strains Xd_1H (1H), Xd_2H (2H) and parental UCD 67–385 (WT) grown on an MM_V agar plate.