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A yeast display library of 5.9 × 10 transformants was created.

The ;;uas-PDFrnai line was created by Dietzl and coworkers [30] and obtained from the Vienna Drosophila RNAi Center (transformant ID: 4380).

The groEL1ΔC mutant was created using the mycobacterial recombineering method [ 9]. pEN15 was digested with AflII and SpeI to create the linear target substrate, which was introduced into mc155 electrocompetent cells, expressing the recombinase genes on pJV53 and in this way creating hygromycin-resistant transformants.

HEK293-LBD cells were created by transfecting HEK293 with pBD-LBD and selecting for Neomycine-resistant transformants.

The Tet-on MnSOD and Cu/ZnSOD expression constructs were created by cloning the corresponding cDNAs [58] into the USC1.0 vector [59] and generating multiple independent transformant strains.

One of the Ura+ transformants was named strain TR2-7.

Gene integration into prototrophic transformants was verified by genomic PCR.

Plasmid DNA from positive transformants was isolated and sequenced.

Thus, for each of the constructs created in this study, 13 to 20 independent transformants were utilized for expression analysis.

The PCR product was transformed into strain AH9, which also contained pKD46 [33], and chloramphenicol-resistant transformants were selected and verified by PCR using primers nin5L 40.410 5'-GTCTTCTGGTTATCGAAGG-3' and nin5R 43.522 5'-CTTTGTCGTAATCGAGATT-3' to create strain SMR6230.

Transformants were confirmed by polymerase chain reaction (PCR) and Southern blots and one correct transformant (TJW123.20) was sexually crossed with RDIT2.1 to create a prototrophic strain (RCSR4.16).