Scheme of TIPI-gTOW. We first constructed a strain in which the chromosomal target gene was replaced by a GFP-
target construct. We next introduced the TEV plasmid, a plasmid for gTOW that encodes pTEV+ expressed from the CUP1 promoter. According to the TIPI procedure, cleavage and rapid degradation of the GFP-TDegFtarget is induced by pTEV+. Using the gTOW procedure, in which the copy number of the TEV plasmid exceeds 100 under the -Leu condition, we can increase the expression of pTEV+, which accelerates the degradation of the GFP-TDegFtarget, reducing the level of the GFP-TDegFtarget. It is thus expected that the upper limit copy number of the TEV plasmid would inversely correlate with the lower limit of the GFP-TDegFtarget. The tug-of-war between the bias to increase the copy number of leu2d and the bias to decrease the copy number of pTEV+ gene determines the plasmid copy number in the cell under the -Leu condition. It is thus possible to indirectly estimate the lower limit of the GFP-TDegFtarget by measuring the copy number of the TEV plasmid.