Fig. 5

(a) Drop-test on solid media determining the minimal inhibitory concentration of geneticin G418 on C. oleaginosus wt. (b) Growth curves of C. oleaginosus wt with increasing concentrations of geneticin G418, determining the minimal inhibitory concentration in liquid media. (c) Map of the pRF_PPIp_APH plasmid, which was used for ATMT-derived integration of the APH resistance gene. Shown elements, besides the described gene cassette, include: KanR, bacterial kanamycin resistance; oriV, bacterial origin of replication; trfA, bacterial plasmid replication initiator protein. Plasmid maps were created with Geneious. (d) Restriped colonies after transformation of the wt control and the transformants on geneticin G418 plates. (e) Gel-electrophoresis of APH amplified by PCR (1599 bp) from genomic DNA of the transformants. The gel was cropped for clarity after lane 5, the complete gel picture is included in Figure S3. (f) Growth curves of the transformants and the wt, in triplicates, with and without the addition of geneticin. APH, aminoglycoside 3’-phosphotransferase; Gen, geneticin; wt, wild type