Fig 3. Rapid phenotypic characterization of a lethal conditional P. falciparum mutant using the plaque assay.

(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 Materials and Methods 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-SERA6 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 SERA6 gene is lethal. Arrow-heads indicate the oligonucleotide primers used for PR analysis: blue, SERA6-34; yellow, JTS5synthF; brown, JTPbDT3’R (see Materials and Methods for primer sequences and PCR parameters). Expected sizes of the PCR amplicons are indicated.