This publication describes one of the methods we have been developing for the needs of the current study funded by GA UK. It enables inducible overexpression of a trypanosome protein of interest from its endogenous locus. Instead of conventional cloning with restriction enzymes, we employed a PCR only-based system. This approach consists of a single-step PCR with a template pPOT plasmid with long forward and reverse primers matching the sequences in the gene coding for the protein of interest. We created a novel pPOT plasmid, which in addition to His6 tag for protein purification and a fluorescence marker introduces also a tetracycline-inducible overexpression cassette. We showed for a number of flagellar and flagellum tip-localizing proteins that the approach can be used to tag and overexpress a range of proteins. In the current study, we used the system to generate the TbRpiB overexpression cell line.