Genomic mini ax yeast spin kit

Yeast strains were transformed using the LiAc/ssDNA/PEG method [160 (link)]. Total DNA was isolated from yeast cultures using the Genomic Mini AX Yeast Spin Kit (A&A Biotechnology, Gdansk, Poland). E. coli cells were transformed as described by Sambrook and Russell [161 ]. Plasmids were isolated from bacteria using the Plasmid Mini Kit (A&A Biotechnology). DNA was extracted from the agarose gel using the Gel-Out Kit (A&A Biotechnology) and purified after enzymatic reactions using the Clean-Up Kit (A&A Biotechnology). Restriction enzymes and DNA ligase (Thermo Scientific, Waltham, MA, USA) were used as recommended by the supplier.

Fusarium culmorum strain JTW1 was isolated using ten-fold dilution procedure on Potato Dextrose Agar (PDA, A&A Biotechnology) from soil samples collected in Różankowo near Toruń, Poland. The Petri plates were incubated at 26°C for 7 days. Initially, the morphological and cultural characteristics of isolate F. culmorum strain JTW1 were studied after 7 days at 26°C on PDA. The isolate was identified by internal transcribed spacer (ITS) sequence. The genomic DNA of the isolate was extracted using Genomic Mini AX Yeast Spin Kit (A&A Biotechnology) according to the manufacturer’s instructions by A&A Biotechnology (Gdańsk, Poland) while amplification of ITS region and sequencing were carried out by Genomed S.A. (Warsaw, Poland). The ITS region of the ribosomal DNA of the isolate was amplified using the ITS1 and ITS2 primers (White et al., 1990 (link)). The Basic Local Alignment Search Tool (BLAST) at the National Centre of Biological Information (NCBI) was used to find the closest similarity between the isolate sequence and corresponding sequences available in the database. F. culmorum isolate JTW1 was deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) in Brunschweig, Germany under accession number DSM 114849.

The restriction enzymes used in this study were purchased from FastDigest Thermo Scientific and the digestions were performed according to the producer’s protocols. The PCR reactions done using Phusion high fidelity DNA polymerase (Thermo Fisher Scientific). Ligation reactions were performed using T4 DNA Ligase (Thermo Fisher Scientific) for 10 min at room temperature. The vectors were isolated using the Plasmid Mini Kit (A&A Biotechnology, Poland). Transformation of E. coli strains was performed using standard chemical protocols. Genomic DNA (gDNA) was isolated from yeast using the Genomic Mini AX Yeast Spin kit (A&A Biotechnology, Poland).

All restriction enzymes were purchased from FastDigest Thermo Scientific (USA), and all of the digestions were performed according to standard protocols. The PCR reactions were set up using recommended conditions and Phusion high-fidelity DNA polymerase (Thermo Scientific). The ligation reactions were performed for 10 min at room temperature using T4 DNA Ligase (Thermo Scientific). The gel extractions were performed using the Gel Out gel extraction kit purchased from A&A Biotechnology (Poland). The E. coli minipreps were performed using the Plasmid Mini Kit (A&A Biotechnology). Transformation of E. coli strains was performed using standard chemical protocols [24 ]. Genomic DNA (gDNA) was extracted from Y. lipolytica using the Genomic Mini AX Yeast Spin kit (A&A Biotechnology).

All restriction enzymes were purchased from FastDigest Thermo Scientific and all of the digestions were performed according to standard protocols. The PCR reactions were set up using recommended conditions and Phusion high-fidelity DNA polymerase (Thermo Scientific). The ligation reactions were performed for 10 min at room temperature using T4 DNA Ligase (Thermo Scientific). The gel extractions were performed using the Gel Out extraction kit purchased from A&A Biotechnology (Poland). The E. coli minipreps were performed using the Plasmid Mini Kit (A&A Biotechnology). Transformation of E. coli strains was performed using standard chemical protocols.
Transformation was performed by the lithium acetate method and transformants were plated out on selective media without uracil as described before (Mirończuk et al., 2016 (link)). They were analyzed for proper integration by gDNA extraction and PCR amplification. Genomic DNA (gDNA) was extracted from Y. lipolytica using the Genomic Mini AX Yeast Spin kit (A&A Biotechnology, Poland).

All of the restriction enzymes, the Phusion high-fidelity DNA polymerase and the T4 DNA ligase were purchased from Thermo Scientific (USA). The reactions followed standard protocols as described by the manufacturers. A Plasmid Mini Kit, Gel Out extraction kit and Genomic Mini AX Yeast Spin kit were obtained from A&A Biotechnology (Gdańsk, Poland). The isolation of plasmid DNA, DNA from gel purification and gDNA extraction followed protocols supplied by the manufacturer.
The E. coli transformation followed the standard chemical protocol with a selective medium containing ampicillin to plate the Y. lipolytica strains, which were transformed with overexpression cassettes or a deletion cassette by the lithium acetate method. The transformations resulted in the strains AJD ΔB07117g and AJD pAD-B07117g. Additionally, the strain AJD ΔB07117 was transformed with an overexpression cassette, resulting in the strain AJD-c-B07117g.

DNA was extracted from dissected bacteriomes or insect abdomens using one of three different DNA extraction kits: Sherlock AX isolation kit (A&A Biotechnology, Poland), Bio-Trace DNA Purification Kit (Eurx, Poland), and Genomic Mini AX Yeast Spin kit (A&A Biotechnology, Poland), according to manufacturers’ protocols. The metagenomic libraries for high-throughput sequencing on the Illumina platform were prepared using NEBNext Ultra II FS DNA Library Prep and NEBNext DNA Ultra II kits, with a target insert length of 350 bp. The pooled libraries were sequenced on Illumina HiSeq X or NovaSeq 6000 S4 lanes (2 × 150 bp reads).