Construction of the pETDuet-1 his-tagged mEhQTRT1-EhQTRTD1 vector.

For the construction of the pETDuet-1 his-tagged mEhQTRT1-EhQTRTD1 having a point mutation of D267 to A267 in EhQTRT1, EhQTRT1 was amplified from E. histolytica genomic DNA using primers standard EhQTRT1 and 1-265-EhQTRT1 XhoI. The second part of EhQTRT1 was amplified using genomic DNA using primers 266-EhQTRT-XhoI (where the aspartate was replaced with alanine in the primer) and reverse primer EhQTRT1. Both PCR products were purified using a QIAquick PCR purification kit (Qiagen), digested with XhoI for 1 h at 37°C, and repurified. The purified products were ligated at 16°C overnight using T4 ligase. Following ligation, the product was amplified by PCR using primers BamHI EhQTRT1 and NotI EhQTRT1. The PCR product was cloned in to the pGEM-T easy vector, digested with BamHI and NotI, and further subcloned into a pETDuet-1 vector that was previously linearized with BamHI and NotI. Following this, the EhQTRTD1 gene was cloned into the pETDuet-1 vector as described above. The resulting plasmid was sequenced to ensure the presence of the desired mutation.