Fig. 1
Overview of plasmid shuffle experimental procedure. Generation of conditional L. donovani NMT null mutants to demonstrate NMT essentiality by plasmid shuffle. Heterozygous NMTHYG/+ promastigotes (1) were episomally transfected with pXNG4-NMT-TK carrying GFP and TK (2), before targeting the 2nd NMT allele for replacement with PAC, creating NMTPAC/HYG (3). Individual clones were then transfected with an episomal plasmid coding for tdtom only or for NMT and tdtom (NMT-tdtom; 4). Ectopic expression of NMT by only one (pXNG4-NMT-TK) of the two plasmids (right hand side scenario) or both (left hand side scenario) will allow genetic validation of its essentiality by negative selection with ganciclovir (GCV, 5). If NMT is an essential gene, mutants will grow, after several passages of negative selection that are only tdtom+, when parasites were transfected with pXNG4-NMT-TK/NMT-tdtom, or GFP+ and tdtom+, when transfected with pXNG4-NMT-TK/tdtom, respectively. The latter may not grow but rather die due to the negative selection. If NMT is non-essential for viability, retention of plasmids during cytokinesis will be subject to a random distribution and tdtom+ and/or GFP+ mutants could arise in both conditional complementation settings or the plasmids will not be retained at all. Histograms shown on the right in 1–4 depict representative fluorescent properties for GFP and tdtom of the heterozygous NMTHYG/+ parasites (1- starting population) and mutants generated by episomal plasmid transfection (2–4, grey filled curve, no fill shows starting population). Abbreviations: NMT, N-myristoyltransferase; SAT, streptothricin acetyltransferase; HYG, hygromycin phosphotransferase; PAC, puromycin N-acetyltransferase; NEO, neomycin phosphotransferase; GFP, green fluorescent protein; tdtom, tandem tomato fluorescent protein; TK, thymidine kinase from Herpes simplex virus; ess, essential