The genomic DNA of P4R5 was extracted and purified using the TIANamp yeast DNA kit (TIANGEN, Beijing, China). Sequencing libraries were generated using NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s protocol. The whole genome of P4R5 was sequenced using Illumina NovaSeq PE150 and assembled with SOAP de novo software at the Beijing Novogene Bioinformatics Technology Co., Ltd. The coding genes were predicted using the Augustus 2.7 program, and the gene function was predicted based on the Non-Redundant Protein Database [45 (link)] and the Kyoto Encyclopedia of Genes and Genomes database. The genome information of P4R5 has been deposited to GenBank under the accession number ID: JAHERV000000000.
Tianamp yeast dna kit
Manufactured by Tiangen Biotech
Sourced in China
The TIANamp Yeast DNA Kit is a DNA extraction kit designed for the isolation of genomic DNA from yeast samples. The kit provides reagents and protocols to efficiently extract and purify high-quality DNA from yeast cells.
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Lab products found in correlation
Sybr green real time pcr master mix plus, by Toyobo (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Novaseq pe150, by Illumina (1 mentions)
Lightcycler 96 real time pcr system, by Roche (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
Qubit 3 fluorometer, by Thermo Fisher Scientific (1 mentions)
Cloneez kit, by GenScript (1 mentions)
Peasy t1 vector, by Transgene (1 mentions)
Abi prism 3100 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Novaseq 6000, by Novogene (1 mentions)
Trizol, by Tiangen Biotech (1 mentions)
Nanodrop one spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Mut express 2 fast mutagenesis kit v2, by Vazyme (1 mentions)
Genome walking kit, by Takara Bio (1 mentions)
One step cloning kit, by Vazyme (1 mentions)
Sybr premix ex taqtm 2 tli rnaseh plus, by Takara Bio (1 mentions)
Lightcycler 480 instrument, by Roche (1 mentions)
E coli dh5α, by Takara Bio (1 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
24 protocols using tianamp yeast dna kit
1
Genomic Analysis of P4R5 Yeast
2
Yeast Genomic DNA Extraction and Phylogenetic Analysis
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Genomic DNA was extracted from a fresh culture of yeast strains followed by TIANamp Yeast DNA Kit (Tiangen Biotech (Beijing) Co., Ltd., Beijing, China) [35 (link),36 (link)] for sequencing and phylogenetic analysis of the 26S rRNA gene and ITS gene. Primers 26S-F (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and 26S-R (5′-GGTCCGTGTTTCAAGACGG-3′) were used to amplify the D1/D2 region [37 (link)]. Primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATAT GC-3′) were used to amplify the ITS gene region [38 (link)]. The amplification protocol [7 (link)] consisted of initial denaturation at 95 °C for 5 min, followed by 31 cycles of amplification at 94 °C for 3 s, 92 °C for 30 s, 40 °C for 1 min, and a final extension at 65 °C for 8 min.
Then amplification products were sequenced by Qingke Biotechnology Co., Ltd., Beijing, China. Sequence similarity was determined using NCBI BLAST (National Center for Biotechnology Information, Bethesda, MD, USA) based on databases—Nucleotide collection (nr/nt), employing Megablast for highly similar sequences. The phylogenetic analysis was conducted by using MEG A-X(64) software after multiple alignments of data via CLUSTAL_X, and the phylogenetic tree was constructed using the neighbor-joining method [39 (link)].
Then amplification products were sequenced by Qingke Biotechnology Co., Ltd., Beijing, China. Sequence similarity was determined using NCBI BLAST (National Center for Biotechnology Information, Bethesda, MD, USA) based on databases—Nucleotide collection (nr/nt), employing Megablast for highly similar sequences. The phylogenetic analysis was conducted by using MEG A-X(64) software after multiple alignments of data via CLUSTAL_X, and the phylogenetic tree was constructed using the neighbor-joining method [39 (link)].
3
Quantifying Antimicrobial Resistance Genes
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Genomic DNA was prepared using the TIANamp Yeast DNA Kit (TIANGEN, Beijing, China). Gene copy numbers of mph and mutants were determined according to the absolute quantification method described by the reference [39] (link), using the SYBR Green Real-time PCR Master Mix-Plus (Toyobo, Osaka, Japan). The primers and quantitative real-time PCR protocol were as described in the previous section.
4
Quantitative PCR for Gene Copy Number
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Primers for qPCR are listed in Additional file 1 : Table S2. Genomic DNA was extracted using the TIANamp Yeast DNA Kit (Tiangen, China) according to the manufacturer’s protocol. Gene copy numbers were determined by quantitative PCR (qPCR) as described by Kolacsek et al. [56 (link)]. The OpMOX and ScALG9 genes were used as the references of O. polymorpha and S. cerevisiae, respectively. The plasmid pWYE3227 harboring partial sequences of gfpmut3a, OpMOX and ScALG9 genes was used as the template for standard curves to estimate the copy number of gfpmut3a. Similarly, to estimate the copy number of cadA, HSA or PScTEF1-TAL-PScTPI1-4CL-PScTEF2-STS expression cassette, the vector pWYE3228 harboring partial sequences of OpMOX, cadA, HSA and TAL was used for standard curves. Quantitative PCR was performed using GoTaq qPCR master mix (Promega, USA) in a 20-μL mixture with a LightCycler® 96 Real-Time PCR System (Roche, Switzerland).
5
Candida albicans DNA Extraction
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The C. albicans suspensions or unlysed blood sediments were heated at 100°C for 15 min and then transferred to −80°C for 10 min (repeated three times). DNA of C. albicans was extracted using the TIANamp Yeast DNA Kit (Tiangen Biotech, Beijing, China). The extracting procedure was performed according to the manufacturer’s recommendations. Briefly, solutions of 200 μL of GA buffer, 20 μL of proteinase K and 220 μL of GB buffer were added to the collected yeast cells and incubated at 70°C for 10 min, and mixed well to lyse the cells adequately. Then, the yeast DNA was precipitated with 220 μL of ethanol and adsorbed with DNA-adsorbing column. After rinsing twice with 70% ethanol solution, elution was performed with 50 µL of Tris-EDTA buffer.
6
Efficient DNA Extraction from CSF and Colonies
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DNA extraction was conducted using glass beads (CapitalBio, Beijing, China) and boiling method as earlier reported with a little revision [26 (link)–28 (link)]. Briefly, a bit of sediments of CSF after centrifugation or fresh colonies were suspended in 50 μl of 1× TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) within an extraction tube and incubated at 95 °C in a boiling water-bath for 5 min. Then the total DNA was isolated by vortexing at maximum speed in an Extractor™ 36 (CapitalBio) for 5 min. After centrifuged at 10000 g for 5 min, the supernatant of the lysate containing gemonic DNA was separated for follow-up tests. This is “glass beads method” for sample cell lysate.
In order to meet the special needs of Fig.4 , we have further purified the supernatant of the lysate with “TIANamp Yeast DNA Kit” (TIANGEN Biotech, Beijing, China) starting from step 7 of the operating manual. This is “spin columns method” for DNA extraction.
DNA concentrations were accurately measured using the “Qubit™ 3 Fluorometer” (Q33216, Thermo Fisher Scientific, Wilmington, USA). All genomic DNA and sample lysate were kept in − 80 °C to preserve, and avoid repeated freeze-thaw cycles.
In order to meet the special needs of Fig.
DNA concentrations were accurately measured using the “Qubit™ 3 Fluorometer” (Q33216, Thermo Fisher Scientific, Wilmington, USA). All genomic DNA and sample lysate were kept in − 80 °C to preserve, and avoid repeated freeze-thaw cycles.
7
P. pastoris gas1 Gene Knockout via Homologous Recombination
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The genomic DNA was isolated from P. pastoris cells using the TIANamp Yeast DNA Kit (Tiangen, Beijing, China). Based on the genome sequence of P. pastoris GS115, DNA fragments upstream and downstream of the open reading frame of gas1 were amplified from genomic DNA by two pairs of primers (gas1-F1 and gas1-R1, gas1-F2 and gas1-R2, respectively). HIS4, a selectable marker for isolating Pichia recombinant strains, was amplified from plasmid pPIC9 using the primers his4-F and his4-R. The three abovementioned DNA fragments were ligated into a DNA fragment named “GH” via overlap-extension PCR with the primers gas1-LF and gas1-RR. In addition, a DNA fragment designated “OA” containing the origin of replication derived from pBR322 and an ampicillin resistance gene was amplified from plasmid pPIC9 with primers OA-F and OA-R. Finally, the two DNA fragments, GH and OA, were ligated into a circular molecule by homologous recombination using the CloneEZ Kit (GenScript, Nanjing, China), generating the recombination vector pGH01. pGH01 was integrated into the gas1 locus of the P. pastoris chromosome via homologous recombination.
8
Phenotypic and Phylogenetic Analysis of Fungal Strain BL06
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The phenotypic analysis of the colonies formed on the PDA plates was performed in accordance with the methods described previously (Jiang et al., 2018 (link); Liu et al., 2020 (link)). The genomic DNA of strain BL06 was extracted using a TIANamp Yeast DNA Kit (TIANGEN, Beijing, China). Amplification and sequencing of the internal transcribed spacer region (ITS) of the rRNA gene cluster were performed using the common primers ITS1 and ITS4 (Supplementary Table S2 ) according to the methods described by Ma et al. (2014) (link). The obtained ITS sequence of strain BL06 was aligned using BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi ). The phylogenetic tree was made in MEGA7.0 by the neighbor-joining method (Kumar et al., 2016 (link); Liu et al., 2020 (link)).
9
Cloning and Sequencing of Lxyl-p1-2
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The genomic DNA of mutants with higher β-xylosidase avtivities was extracted via TIANamp Yeast DNA Kit (TIANGEN, Beijing, China) and used as the PCR template. The standard primer 5′AOX1 and 3′AOX1 were used to amplify the Lxyl-p1–2 variants. Then the amplified fragment was cloned into the pEASY-T1 vector (Transgen, Beijing, China). It was then sequenced using a DNA sequencer (ABI PRISM® 3100 Genetic Analyzer, Applied biosystems, Foster City, CA, USA). The DNA sequences were analyzed compared with the wild type parent using the DNAMAN software, (Version 6.0, Lynnon Corporation, San Ramon, CA, USA).
10
Screening Yeast Transformants by Colony PCR
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Transformants were grown on a solid EMM plate at 30 °C for 3 days. Four individual colonies were then selected, followed by overnight culture in YE5S for yeast proliferation. Yeast genomic DNA was obtained using the TIANamp Yeast DNA Kit (TIANGEN, Beijing, China). Colony PCR was conducted by using the primers F_nmt and R_GFP, as listed in Table 1 , and the respective transformant genomic DNA as the template, where SPBPJ4664.02Δ was used as the negative control.
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