Nebuilder hifi dna assembly reaction mix

Genomic DNA of strain ESM356 (wild-type) was isolated using a lithium acetate-SDS based protocol as previously described (Looke et al., 2011 (link)). PCR amplification of the desired fragments was performed using NEB Q5 High-Fidelity DNA polymerase (#M0491S, NEB). The PCR fragments were assembled into the linearized vector pRS305 (integration vector; Sikorski and Hieter, 1989 (link)) using NEBuilder HiFi DNA Assembly reaction mix (#E2621L, NEB) as per the manufacturer's instructions. The reaction product was transformed into Escherichia coli TOP10 cells (#C404010, Invitrogen) and plasmid isolation was performed using the Thermo Fisher GeneJet Miniprep Kit (#K0503, ThermoScientific). The positive transformants were confirmed using restriction digestion and sequencing. ESM356 (S288c background) was used as a wild-type strain and all subsequent strains were derived from it. Yeast culturing and transformation were undertaken using established protocols. Yeast expression plasmids carrying mNG–Tpm1 and –Tpm2 (piSP347 and piSP349, respectively) were linearized with the KasI restriction enzyme and transformed for integration into the leu2 locus in the yeast genome as a second copy. The positive transformants were selected on SC-Leu plates and expression of mNG–Tpm1 and –Tpm2 were confirmed using fluorescence microscopy.

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.