Fig. 2. Disruption constructs targeting mhcA and amiA, and confirmation of disruption using genomic PCR. (A) The targeting vector used to knockout amiA was constructed by replacing part of its coding region and promoter with the blasticidin S [pKO amiA(Bsr)] or G418 [pKO amiA(Neo)] resistance gene. In the targeting vector for mhcA, a portion of the motor domain was replaced with the G418 resistance cassette. Expression of all drug resistance genes was driven by the actin 15 promoter. Thick lines indicate coding sequences of amiA and mhcA. (B) Mutant cells were identified by a shift in size of the PCR products. (Left) Knockout of amiA in mhcA^- cells (HS1), yielding HTU1; (middle) knockout of amiA in corA^- cells, yielding HTU8; (right) knockout of corA in mhcA^- cells (HS1), yielding HTU7. Arrows in A show the positions of the primers used for genomic PCR.

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