FastDigest SalI

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	The 3 DNA fragments were digested with 4 kinds of FastDigest restriction enzymes (KpnI, HindIII, BamHI, and SalI) in FastDigest Buffer (Thermo Fisher Scientific, Waltham, MA, USA), and ligated to the KpnI-SalI sites of the pUC19 vector (Takara Bio) to yield a plasmid, pAYA1, carrying the ΔrodZ::hph mutation.

Protocol tips
The 3 DNA fragments were digested with 4 kinds of FastDigest restriction enzymes (KpnI, HindIII, BamHI, and SalI) in FastDigest Buffer (Thermo Fisher Scientific, Waltham, MA, USA), and ligated to the KpnI-SalI sites of the pUC19 vector (Takara Bio) to yield a plasmid, pAYA1, carrying the ΔrodZ::hph mutation.

Publication protocol

Gene disruption targeting D. grandis rodZ was performed using a method that was originally developed to generate deletion mutants in Deinococcus radiodurans [20], with modifications. A 769-bp DNA fragment upstream of the rodZ promoter region and a 770-bp DNA fragment downstream of the rodZ open reading frame were amplified via PCR using D. grandis genomic DNA and oligonucleotide primer sets (Table 1). For PCR reaction, Tks Gflex DNA polymerase (Takara Bio, Shiga, Japan) was used. A 1,289-bp DNA fragment (KatHPH cassette) containing the D. radiodurans katA promoter and the E. coli hygromycin-resistance gene (hph) from pKatHPH4 [21] was also amplified by PCR using the oligonucleotide primer set pKat-FP and pKatRP (Table 1). The 3 DNA fragments were digested with 4 kinds of FastDigest restriction enzymes (KpnI, HindIII, BamHI, and SalI) in FastDigest Buffer (Thermo Fisher Scientific, Waltham, MA, USA), and ligated to the KpnI-SalI sites of the pUC19 vector (Takara Bio) to yield a plasmid, pAYA1, carrying the ΔrodZ::hph mutation. A 2,752-bp DNA fragment containing the ΔrodZ::hph mutation was amplified from pAYA1 via PCR using the pKat-FP/pKatRP oligonucleotide primer set and introduced into the D. grandis wild-type genome.

Transformation of D. grandis was performed as follows. D. grandis cells (1 ml) cultured at 30 °C for 24 h were washed with 1 ml of TGY broth and resuspended in 0.1 ml of TGY broth. The cell suspension was mixed with 40 µl of 0.3 M CaCl2. A 30 µl aliquot of the cell suspension was mixed with 5 µl of DNA and incubated at 30 °C. After 90 min, 2 ml of TGY broth was added to the mixture and cultured at 30 °C. Following 24 h, cells were harvested via centrifugation and resuspended in 0.4 ml of TGY broth. Aliquots of 0.1 ml were spread on TGY agar plates supplemented with 50 µg/ml hygromycin B (FUJIFILM Wako Pure Chemical) and incubated for 2 to 3 d until colonies of transformants appeared on the plate. A single colony was diluted and spread again on TGY agar plate supplemented with 50 µg/ml hygromycin B for pure culture. The resultant strain was designated ΔrodZ.

The D. grandis genomic DNA was isolated using a FastDNA Spin Kit with a FastPrep-24 Instrument (MP Biomedicals, Santa Ana, CA, USA). Gene disruption was confirmed by amplifying the target allele by genomic PCR using the oligonucleotide primer set HpH-FP and HpH-RP (Table 1)."

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