Generation of CYC17 knockout strains

To construct CYC17 knockout strains (cyc17Δ), a gene knockout sequence was cloned. For this, ∼1 kb upstream and downstream DNA sequences of the CYC17 ORF were amplified using the following primers: upstream, CYC17-5f652-NotI 5′-AGTTCTAGAGCGGCCGCTAGATATTTTGGGTAGATAAGACAC-3′ and CYC17-5r1589-N4 5′-GTCAGGTGCCTGGTACCCTGCAATATTAAAATAAATAAAAATG-3′; downstream, CYC17-3f3368-N4 5′-CTGACGTCGCACCATCCCTTTTTTACTAAGACCTTAATTTATC-3′ and CYC17-3r4316-NotI 5′-ACCGCGGTGGCGGCCGCTCTATCATATTCAGTTTGAGGAG-3′. The 2 sequences were then assembled into a NEO4 cassette containing a Tetrahymena codon optimized neomycin resistance gene under the control of a Cd²⁺-inducible metallothionein (MTT1) promoter using fusion PCR, and cloned into the pBlueScript SK (+) vector. The knockout fragment was released by NotI digestion and transformed into starved CU427 (VI) and CU428 (VII) WT cells using a biolistic transformation. The transformants were selected using SPP medium containing CdCl₂ (1 μg/ml at the start of selection and 0.05 μg/ml at the end) and increasing concentrations of paromomycin (from 120 μg/ml to 40 mg/ml) until all WT chromosomes in the MAC were replaced by knockout chromosomes.⁴²