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. 2018 Dec 5;20(3):447–459. doi: 10.1111/mpp.12766

Figure 4.

Figure 4

The Tef::URA3B cassette was removed by homologous recombination and selected by 5‐fluoroorotic acid (5‐FOA) treatment. (a) Schematic diagrams of the removal of the Tef::URA3B cassette (Cob_09513). Green arrows show the homologous 500‐bp sequences for recombination. (b) Genomic DNA polymerase chain reaction (PCR) showed that Tef::URA3B was removed. The primer set Po13/Po14 generates the 1200‐bp amplicon if the Tef::URA3B cassette is present. The 266‐bp bands corresponding to CHITIN SYNTHASE (CHS) were amplified using the primer set CHS_79F and CHS_345R (Carbone and Kohn, 1999) to show the presence of genomic DNA. The primers used are listed in Table S3 (see Supporting Information). (c) ura3a/b#1, pks1/ura3a/b‐Tef::URA3B#1 and pks1/ura3a/b#1‐4 strains were cultured on potato dextrose agar (PDA), PDA with 10 mm uridine and PDA with 10 mm uridine plus 1 mg/mL 5‐FOA for 6 days at 25 °C in the dark. All pks1/ura3a/b#1‐4 strains showed uridine auxotrophy and 5‐FOA insensitivity similar to ura3a/b#1, demonstrating successful removal of the Tef::URA3B cassette. The details of each strain are listed in Table S1 (see Supporting Information). [Colour figure can be viewed at wileyonlinelibrary.com]