Site-Directed Mutagenesis of nrdJ Gene

The introduction of mutations for targeted amino acid exchange was performed by Ω-PCR with the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies). As a template, the expression plasmids pET28b(+) were applied, containing the respective nrdJ gene between the restriction sites NdeI and HindIII (16 , 17 (link)). The mutagenesis was performed according to the manufacturer's instructions from the QuikChange II Site-Directed Mutagenesis Kit. The mutagenesis product was transformed into electrocompetent Escherichia coli TOP10. The identity of the variant was confirmed by plasmid preparation (Miniprep kit, Macherey-Nagel GmbH & Co. KG) and sequencing (Microsynth Seqlab GmbH). Plasmids with the confirmed mutation were transformed into E. coli BL21(DE3) for recombinant gene expression.