Rapid phenotypic characterization of a lethal conditional <i>P</i>. <i>falciparum</i> mutant using the plaque assay.

<p>(A) Left hand-side; schematic of the results of plaque analysis of RAP-treated and DMSO (mock)-treated SERA6:loxP parasites. Microplate wells coloured green indicate those that contained plaques 14 days following plating out the parasites at a theoretical 10 parasites/well. White wells contained no plaques (wells shown in grey were not used for the cloning). Whereas plaques were present in every well of the mock-treated culture, only a single plaque appeared in one well (well D8) of the RAP-treated culture. Right hand-side; example wells from the RAP-treated and control plates (green channel only of the scanned image shown to enhance plaque visibility, displayed as a grayscale image; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157873#sec008" target="_blank">Materials and Methods</a> for details). (B) Diagnostic PCR analysis of either the bulk SERA6:loxP parasite population immediately following RAP or DMSO-treatment (before plaque assay), or parasites expanded from well D8 of the +RAP plate. RAP-treatment significantly reduced the intact-<i>SERA6</i> locus-specific signal in the parasite population and resulted in appearance of a signal specific for the excised locus. Parasites rescued from well D8 of the RAP-treated parasites displayed a non-excised genomic architecture. The results strongly suggest that excision of the <i>SERA6</i> gene is lethal. Arrow-heads indicate the oligonucleotide primers used for PR analysis: blue, SERA6-34; yellow, JTS5synthF; brown, JTPbDT3’R (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157873#sec008" target="_blank">Materials and Methods</a> for primer sequences and PCR parameters). Expected sizes of the PCR amplicons are indicated.</p

Phenotypic characterisation of an MSP1 mutant using the plaque assay.

<p>Scatter plots showing the distribution of plaque sizes obtained following treatment of MSP1:loxPint parasites with DMSO (control, mock-treated) or RAP to induce DiCre-mediated truncation of MSP1. Plaque numbers (n = 205 for the DMSO-treated samples and n = 54 for the RAP-treated samples) were counted manually. Plaque dimensions were quantified using the Magic Wand tool of Photoshop CS5 (Adobe). Horizontal bars indicate mean plaque area ± 1 SD.</p

Poisson analysis of plaque formation shows that each plaque derives from a single parasite-infected erythrocyte.

<p>Poisson analysis of plaque formation shows that each plaque derives from a single parasite-infected erythrocyte.</p