Agarose gel dna purification kit ver 2

pINA1296/pro-TGase and pINA1297/pro-TGase vectors were digested with Not I (Fig. 1) and the resulting DNA fragments carrying the pro-TGase gene were recovered using the Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa, Dalian, China). Not I digestion linearizes the pINA1296/pro-TGase vector in the pBR322 region, allowing targeting its integration into the pBR322 docking platform of the Po1g strain [14 (link)]. In contrast, in the pINA1297/pro-TGase vector, Not I digestion liberates a “yeast cassette” devoid of bacterial DNA, bordered by zeta sequences and containing the ura3d4 defective selection marker. Zeta sequences are LTRs (long terminal repeats) of Ylt1 retrotransposon and have the property to promote non-homologous integration into the genome of Po1h strain [14 (link)]. The defective ura3d4 allele is required in multiple copies to complement the auxotrophy of the Po1h host, which leads to an amplification of the copy number of “yeast cassette” (hp4d::XPR2pre::pro-TGase:: XPR2term) integrated into the host genome [14 (link)]. The method described by Xuan et al. [28 (link)] was used for the transformation of Y. lipolytica, with selection on YNB-N₅₀₀₀ (1.7 g/L yeast nitrogen base without amino acids and ammonium sulfate, 10 g/L glucose, and 5 g/L ammonium sulfate) minimal medium plates.

The cDNA sequence of AwMyD88 was obtained by reverse transcription - polymerase chain reaction (RT-PCR). Gene specific primers (Supplementary Table S1) were designed using Primer Premier 5.0 software according to the sequences of the AwMyD88 gene from our constructed A. woodiana cDNA library. The open reading frame (ORF) sequence of AwMyD88 was amplified using LA Taq DNA polymerase (Takara) in a 25 μl reaction volume containing 15.25 µl of dH₂O, 4 µl of dNTP Mixture (2.5 mM each), 2.5 µl of 10×LA Taq Buffer II (Mg²⁺ Plus), 1 µl of each primer (10 μM), 0.25 µl of LA Taq and 1 µl of cDNA template. The PCR amplification program was performed according to the manufacturer’s protocol. Briefly, 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 2 min were conducted for amplification. The PCR amplification products were separated using 1.0% agarose gel electrophoresis with Goldview nucleic acid stain, purified with a TaKaRa Agarose Gel DNA Purification Kit Ver.2.0 and subsequently cloned into the pMD19-T plasmid vector (Takara, Japan) according to the manufacturer’s instructions. Positive colonies were screened and further confirmed by DNA sequencing.

PCR products were purified with the Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa), and were directly sequenced by Sangon Biotech Co., Ltd. (Shanghai, China) or ligated into pMD18-T Vector or pMD19-T Simple Vector (TaKaRa). Then, the recombinant plasmids were transformed into E. coli DH5α cells according to TaKaRa’s guidelines, and were sequenced by Sangon Biotech Co., Ltd.