2 2 bipyridyl iron chelator

Constructs for designing recombinant proteins were defined by aligning TgNFS2 and TgISU1 amino acid sequences with their E. coli counterparts. For TgNFS2, a 1,438 bp fragment corresponding to amino acids 271–699, was amplified by polymerase chain reaction (PCR) from T. gondii cDNA using primers ML4201/ML4012 (sequences of the primers used in this study are found in S6 Table). For TgISU1, a 393 bp fragment corresponding to amino acids 64–194, was amplified by PCR from T. gondii cDNA using primers ML4204/ML4205. The fragments were cloned into the pUC19 (Thermo Fisher Scientific) using the HindIII/BamHI and SphI/BamHI restriction sites, respectively. E. coli mutants from the Keio collection (obtained from the The Coli Genetic Stock Center at the University of Yale: stain numbers JW1670-1 for SufS, JW2513-1 for IscU), were transformed with plasmids for expressing recombinant TgNFS2 and TgISU1 and selected with ampicillin. For growth assays [41 (link)], overnight stationary phase cultures were adjusted to the same starting OD₆₀₀ of 0.6 in salt-supplemented M9 minimal media containing 0.4% glucose and varying amounts of the 2,2′-Bipyridyl iron chelator (Sigma-Aldrich). Parental E. coli (strain K12) were included as a control. Growth was monitored through OD₆₀₀ measurement after 7, 14 and 24 hours at 37°C in a shaking incubator.