Full length iscu, mpgm, and pss P. canceri genes were commercially synthesized in pUC57 plasmids (Genewiz). The pDDGFP_LEUD plasmid (Addgene plasmid #58352) was used for expressing recombinant proteins in Saccharomyces cerevisiae (Parker and Newstead 2014 (link)). Plasmids containing genes of interest and the pDDGPD_LEU2D destination plasmid were recovered using the Nucleospin plasmid DNA purification kit (Macherey-Nagel) following the protocol provided by the manufacturer. Plasmids were linearized with 12 U/µl of SmaI (Promega) at 25 °C for 1 h. SmaI was inactivated at 70 °C for 15 min. Linearized plasmids were digested with 10 U/µl of BamHI (Promega) at 37 °C for 1 h and recipient pDDGPD_LEU2D was dephosphorylated with 1 µl of Thermosensitive Alkaline Phosphatase (TSAP, Promega) at 37 °C during the last 15 min of incubation with BamHI. Finally, TSAP and BamHI were inactivated at 74 °C for 15 min. Digestion products were ligated into dephosphorylated recipient plasmids at a 1:2 vector:insert ratio with 3 U of T4 DNA ligase (Promega) and at room temperature for 15 min according to the protocol of the manufacturer. Ligase was inactivated at 70 °C for 10 min. Exact protocols available upon request from the authors. P. canceri genes were cloned upstream of green fluorescent protein (gfp) encoded on the pDDGFP-LEU2D plasmid and transformed into Escherichia coli DH5α by standard methods and selected on synthetic defined media (LB plus 50 ug/ml ampicillin). Polymerase chain reactions (PCR) were performed to screen for colonies with gene insert using Gotaq Green master mix (Promega) and 10 µM of each primer (Forward primer: gal1 5′”-TCTGGGGTAATTAATCAGCGAAGCG-3′”. Reverse primers: iscu 5′”-GCTCCCGCAGCCGAAAGTCTTAAA −3′”, pss 5′”-AGGATGCGGCCTACTGAATGGACC-3′”, mpgm 5′”- GGGGCATAGGATCCATGACTCAATGGTGTGTA-3′” and gfp 5′”-AGTAGCGTCACCTTCACCTTCACC-3′”) using standard thermocycling conditions (95 °C 5 min; 30 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, final elongation temperature of 72 °C for 10 min). The insertion of P. canceri genes into the pDDGFP_LEU2D vector was confirmed by Sanger sequencing with gal1_forward and gfp_reverse primers (Eurofins).
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