FIGURE 3.

PKH_020910 is essential in P. knowlesi. A, outline of the transfection strategy and location of the primers used for the verification of integration of the plasmids. The plasmid containing the fragment of PKH_020910 was linearized with BsaBI within the PKH_020910 coding sequence to promote recombination and prevent plasmid propagation. Note that the 5′ region of PKH_020910 that encodes the signal sequence was omitted from the integration region. The duplicated copy of PKH_020910 produced following integration therefore lacks a start codon and cannot give rise to an exported protein. The plasmid backbone is shown in purple. B, verification of integration of the PKH_020910 targeting plasmids using the primers shown in A. Integration-specific products were detected only with DNA from parasites transfected with targeting vectors that would reconstitute the entire gene (PKH_020910 476 and PKH_020910 428+recodonized) and not with the targeting vector designed to truncate the gene to produce a non-functional protein or with DNA from untransfected parasites. The number after the gene name indicates the last codon of PKH_020910 in the targeting region. Expected sizes of the PCR products are as follows: 1+2,1469 bp; 3+4, 1919 bp; 1+5, 1453 bp; 1+4, 1892 bp. Sizes of relevant standards are indicated on the left-hand side. All transfections were repeated a minimum of three times, each time yielding the same result.