Fig 3. Generation of heterozygous mutants of LdThrRS.

(A) Map of LdThrRS genomic locus and pSP72α-zeo-α-ThrRS episomal construct is shown with the position of the primers used for confirmation of WT and mutant parasites by PCR-based analysis along with the expected band sizes. Primer pairs are shown in Table 2. Primer 4 was designed as a forward primer to match the upstream region of LdThrRS gene, and primers 8, 3 and 6 were designed internal to LdThrRS, HYG and NEO coding regions, respectively. Primer 2 was designed as a reverse primer to match the downstream region of LdThrRS gene and primers 7, 1 and 5 were designed as forward primers, internal to LdThrRS, HYG and NEO coding regions, respectively. Forward primer Zeo^FP and reverse primer Zeo^RP were designed to match the upstream and downstream region of zeocin resistance gene. Genomic DNA from WT, heterozygous parasites (ThrRS/NEO or ThrRS/HYG) was used as a template for PCR analysis. (B) The specific fusion of the replacement cassette(s) was checked with NEO and gene-specific primers as reported in Table 2 and Fig 3A. Lane 1 (Primers 4 and 6); Lane 2 (Primers 5 and 2); Lane 3 (Primers 4 and 8) and Lane 4 (Primers 7 and 2). (C) The specific fusion of the replacement cassette(s) was checked with HYG and gene-specific primers. Lane 1 (Primers 4 and 3); Lane 2 (Primers 1 and 2); Lane 3 (Primers 4 and 8) and Lane 4 (Primers 7 and 2). (D) WT genomic DNA was used as a negative control. The bands corresponding to the WT gene were obtained in Lane 3 and 4. No bands were observed after using NEO specific primers, Lane 1 (Primers 4 and 6); Lane 2 (Primers 5 and 2) and HYG specific primers, Lane 5 (Primers 4 and 3); Lane 6 (Primers 1 and 2). M indicates the molecular size marker in kb. (E) Southern blot analysis of genomic DNA from wild-type (WT) (Lane 1), ThrRS/NEO (Lane 2) and ThrRS/HYG (Lane 3) parasites. Genomic DNA from WT, ThrRS/NEO and ThrRS/HYG parasites were digested with SphI, separated on a 0.6% agarose gel and probed with 3’UTR of LdThrRS gene. (F) PCR analysis of rescue mutants (ThrRS/NEO[pThrRS⁺]). The specificity of recombination was checked with Zeo^r specific primers, Lane 1 (Primers Zeo^FP and Zeo^RP). M indicates the DNA molecular size marker. (G) Western blot analysis of WT (Lane 1), heterozygous parasites (ThrRS/NEO) (Lane 2), ThrRS overexpressors (WT[pThrRS⁺]) (Lane 3) and rescue mutant (ThrRS/NEO[pThrRS⁺]) (Lane 4) parasites. β- tubulin was used as a loading control.