The sir2Δ fob1Δ double mutant was constructed by transforming UCC8832 with plasmid pRS314-SIR2 [65] to complement the mating defect of sir2Δ, followed by mating to UCC524 and sporulation. Because both deletions are marked with KANMX, PCR was used to identify double mutant spore products to generate UCC8839 and UCC8840. These haploids were mated to generate UCC8844, which was subsequently cured of the pRS314-SIR2 plasmid. Standard mating and sporulation, followed by PCR to identify double mutant spore products, was used to generate csm1Δ fob1Δ and lrs4Δ fob1Δ double mutant strains.
