Heterologous expression StnA, StnB and StnC

The single subunits of the Stn complex were amplified using Phusion DNA polymerase (New England BioLabs; Ipswitch, MA) and genomic DNA of S. ovata as template. Primers used were: 5′-tttcatatgtggagccacccgcagttcgaaaaatctgcgtgtaattcgtgcgaaaaagagct-3′ and 5′-tttggatccctaaccttcacttacaacaccgcctttc-3′ for stnA, 5′-tttcatatgtggagccacccgcagttcgaaaaatctgcggtgaaggttagagtaggtcttgg-3′ and 5′-tttggatccctactcgatacaaactgcgtctaat-3′ for stnB and 5′-tttcatatgtggagccacccgcagttcgaaaaatctgcgagcaaaattagtataaatataaatggccgc-3′ and 5′-tttggatccctacaaactctgcctaccatttacaaaattc-3′ for stnC. The PCR products were cloned into the NdeI and BamHI restriction sites of pET21a and subsequently, the constructs were used to transform E. coli HB101. Plasmids were verified by DNA sequencing and introduced to E. coli BL21 (DE3) ΔiscR, which was already used for the successful production of iron sulfur-cluster containing proteins. Production and purification of the Stn subunits was performed as described earlier³⁵.