Wizard plus sv miniprep kit

In order to amplify the knockout fragment containing approximately 700 bp upstream and downstream region of targeted gene, flanking primers with 15-18 bp overlap (Table S1) were designed. The upstream and downstream regions were first amplified using the high-fidelity Platinum SuperFi PCR master mix (Invitrogen) and purified from a 1% agarose gel (QiAquick gel extraction kit, Qiagen). The fragments were then spliced together by overlap extension PCR and gel purified. Plasmids were isolated from overnight cultures using Wizard Plus SV miniprep kit (Promega). Plasmid and the knockout fragment were digested using SpeI-HF or BamHI-HF (New England Biolabs) for 2 h at 37 °C and gel purified. The restriction digested knockout fragment and plasmid were ligated together using T4 DNA ligase (New England Biolabs) and transformed into E. coli Jke201 using the heat-shock method. After a heat shock, the cells were resuspended in pre-warmed LB broth supplemented with 100µM DAP and incubated at 37 °C for 2 h before plating on an ampicillin (100 µg/ml) selection plate. Transformants were screened by colony PCR using the primers flanking the upstream and downstream regions of the targeted gene (Table S1).

Yeast containing bait and plasmid were plated on SD galactose (‐His/‐Trp/‐Leu) to induce gene expression and select for bait–prey interactions. After incubating at 28°C for ~5 days, colonies were pooled in 10 ml of sorbitol/phosphate buffer (1.2 M sorbitol, 0.1 M NaPO₄, pH 7.5) per plate, pelleted, and resuspended in 2 ml of sorbitol/phosphate buffer supplemented with 500 U of lyticase (Sigma: L2524‐25KU) and 250 μg of RNase A. Yeast cells were incubated in the lyticase buffer for 3 hr at 37°C prior to plasmid recovery. Plasmid DNA was extracted using a Wizard Plus SV Miniprep kit (Promega) and a modified protocol. Briefly, 2.5 ml of lysis solution and 80 μl of alkaline protease solution were added to yeast protoplasts and incubated at room temperature for 10 min. Next, 3.5 ml of neutralization solution was added and cellular debris was pelleted by centrifugation. Supernatant was run through the provided columns and plasmid DNA eluted according to the manufacturer's instructions. Low‐cycle PCR was performed to amplify cDNA's from the prey library. Briefly, MyFi™ proofreading DNA polymerase (Bioline) and pB42AD forward and reverse primers (flanking the cDNA insertion site of pB42AD) were used to amplify cDNA's (Supporting information Table S1). A QIAquick PCR purification kit (Qiagen) was used to clean PCR products before sequencing.