Q5 high fidelity dna polymerase master mix

Genomic DNA was extracted from cells and tissue using DNeasy Blood & Tissue Kit (QIAGEN, Germany). The region of expected indel formation was amplified by PCR asymmetrically spanning the region using the Q5 High-Fidelity DNA polymerase master mix (New England BioLabs, MA, USA) with primers shown in Supplemental Information. The PCR product was denatured and reannealed before cleavage with T7 endonuclease 1 (New England BioLabs, MA, USA) according to the manufacturer’s instructions. Fragments were analyzed by a DNA 1000 assay on an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA) with indel formation frequency determined as per Ran et al.⁵⁵ (link)

The 2-hydroxyacyl-CoA lyase gene (WP_018331913.1) was cloned into expression vector pASG-IBA43 according to the manufacturer’s protocol (IBA Lifesciences) after amplification using the Q5 High-Fidelity DNA Polymerase Master Mix (New England Biolabs), genomic DNA from strain DSM 45062, forward primer 5′-AGC GGC TCT TCA ATG GCG GAC CGG CAG GAC-3′ and reverse primer 5′-AGC GGC TCT TCT CCC GAT CCC TTC CTG ACG GCG-3′. The PCR conditions were initial denaturation of 3 min at 94°C, 25 cycles of 30 s 94°C, 45 s 60°C and 2 min 72°C, and a final elongation of 5 min at 72°C. Chemically competent cells of strain E. coli BL21 (DE3) were transformed with the constructed lyase-bearing vector via heat shock at 42°C. Heterologous expression of the lyase in lysogeny broth supplemented with 100 mg L^–1 ampicillin was induced at 30°C for 5 to 7 hours by 200 μg L^–1 anhydrotetracycline after an initial growth at 30°C from an optical density at 550 nm of 0.1 to a value of 0.6. Cells were harvested by centrifugation and mechanically disrupted with glass beads (Yaneva et al., 2012 (link)) in phosphate buffer (50 mM potassium phosphate, pH 7.2).