Frozen ez yeast transformation 2

Yeast two-hybrid analyses were performed using the matchmaker LexA system following the manufacturer’s manual (ClontechYeast protocol No. PT3024–1). The coding sequence (CDS) of TGNap1 was cloned into pGilda-GW bait vector and the CDSs of Rab6, YIP4A and YIPB were cloned into pB42AD-GW prey vectors using LR reaction (Invitrogen, USA) individually to test the interactions. For the YIPs, the N-cytosolic tail (aa 1–140) of YIP4A ((YIP4B(-TM)) or (aa 1–143) for YIP4B ((YIP4B(-TM)) were used. The CDS of RAB6 was cloned also in pGilda to test the interaction with YIP4A and YIP4B, primers are listed in Supplementary Table 1. Each bait-prey combination was co-transformed into yeast strain EGY48. Competent cell preparation and transformation were performed using the Frozen EZ Yeast Transformation II (Zymo Research T2001). Protein–protein interactions were tested by β-galactosidase assay. The plate pictures were taken after 3 days of incubation at 28 °C. For the assays, positive or negative controls were coexpression of pLexA-53 and pB42AD-T vectors or empty plasmid, respectively, (pGilda empty and pB42AD empty). For each bait and prey combination, the assays were repeated three times with consistent results.

Expression plasmids encoding GAL4 DB (DNA-binding domain) and AD (activation domain) fusion proteins were transformed into the Saccharomyces cerevisiae strains PJ69-2A(MATa) and Y187(MATα) respectively, using the Frozen-EZ Yeast Transformation II (Zymo Research), and plated out on to suitable drop-out medium, according to the manufacturer′s protocol. The yeast-mating procedure and semi-quantitative β-galactosidase assay were performed as described previously14 (link).

Upstream and downstream 1-kb nucleic acid fragments of the CH_4347 encoding frame in strain BMU05228 were amplified by PCR and ligated to the ends of an AMP-HPH fusion accordingly with NEBuilder HiFi DNA assembly reaction mix (NEB, USA). The AMP and HPH gene fragments were amplified from pYes2 and pAN-7 plasmid (Invitrogen, USA), respectively. The ligation product was transformed into Escherichia coli competent cells. Colony PCR and subsequent plasmid extraction were performed to confirm the successful construction and transformation of the ligated vector. The linearized vector with CH_4347 flanking nucleic acid fragments was further transformed into BMU05228 competent cells by the use of Frozen-EZ Yeast Transformation II (Zymo Research, Canada). Peptone-dextrose agar (PDA) plates with 100 μg/ml hygromycin B were used for screening of fungi successfully transformed with the recombinant plasmid, and colony PCR was performed to screen the fungi in which recombination had happened. PCR was performed to amplify the fragment spanning the CH_4347 encoding frame, and the product was sequenced to validate the successful knockout of CH_4347 in BMU05228.