Gel extraction kit

CIDEA WT and point-mutation (R47E and R171E) constructs were previously generated as detailed in Barneda et al.^{27 (link)}. Insertion fragments were amplified by PCR (list of primers can be found in Supplementary Data 1), purified by PCR purification kit (Roche, 11732676001), followed by restriction enzyme digestion. Digested fragments with sticky ends were purified by gel electrophoresis and extracted by gel extraction kit (NEB, T1020S). Purified inserts were ligated to pre-digested pPyCAGIP plasmids by DNA ligase (NEB, M020S) at 16 °C overnight. Ligation product was transformed into DHFalpha competent bacterial cells and amplified. Bacteria culture was lysed, and plasmids purified by midiprep plasmid extraction kits (Invitrogen, K210004), followed by Sanger sequencing for verification.

DNA fragments containing P_ydiU, P_hyaA, or their mutant derivatives were isolated from the relevant plasmids following digestion with BamHI and HindIII. The 3′ end of the top strand was radiolabeled with [α-³²P]dGTP (PerkinElmer) using Klenow fragment. The radiolabeled fragment was isolated from a 5% acrylamide gel, purified using a QIAquick gel extraction kit, and incubated with either 100 nM E. coli RNA polymerase with σ⁷⁰ (NEB), 500 nM purified WT IscR, or both for 30 min at 37°C in a solution containing 40 mM Tris (pH 7.9), 30 mM KCl, 100 μg/ml BSA, and 1 mM DTT. A total of 2 μg/ml of DNase I (Worthington) was added to the reaction mix for 30 s, followed by the addition of sodium acetate and EDTA to final concentrations of 300 and 20 mM, respectively, to terminate the reaction. The reaction mix was ethanol precipitated, resuspended in urea loading dye, heated for 60 s at 90°C, and loaded onto a 7 M urea–8% polyacrylamide gel in 0.5× TBE buffer. An A+G ladder was made by formic acid modification of the radiolabeled DNA, followed by piperidine cleavage (53 (link)). The gels were visualized using a Typhoon FLA-9000 gel imaging scanner.

After the amplification of the msp1 gene, P. falciparum were sorted out to exclude multiclonal infection since multiple clonal infections produce multiple products of pfcsp, which later give non-specific sequencing results. Genomic DNA of single clonal infections were further used for the amplification of pfcsp. The pfcsp gene amplification was done as previously published by Zeeshan et al. [9 (link)]; using the following primers; outer PfCSP-f: 5′ TTA GCT ATT TTA TCT GTT TCT TC 3′ and outer PfCSP-r: 5′ TAA GGA ACA AGA AGG ATA ATA CC 3′, for the first PCR reaction. The second PCR reaction was made using the primers; nested nPfCSP-f: 5′ GAA ATG AAT TAT TAT GGG AAA CAG 3′ and nPfCSP-r: 5′ GAA GGA TAA TAC CAT TAT TAA TCC 3′. PCR amplicons of the first run were used as a template for the second PCR. The amplified pfcsp products were visualized using agarose gel electrophoresis in comparison to a 100 base pair DNA ladder. Amplified PfCSP PCR products were further purified from gel using gel extraction kits and instructions were followed according to the manufacturer’s guidelines (New England Biolabs Inc, New England). Each amplicon was sent in replicate for sequencing by the Sanger dideoxy sequencing method using the primer SeqPfCSP: 5′ TGG GTC ATT TGG CAT ATT GTG 3′ using ABI3500 sequencer (Applied Biosystems SeqStudio, 3500 series) provided by Macrogen Inc. (The Netherlands).