(A) Outline of the genetic tug-of-war method. The explanation of the method is described below. (B) Structure of the plasmid vector (pTOW40836) and the GFP-op plasmid (pTOW40836-TDH3pro-GFP) used in this study. In the case of tGFP-op and NETS-tGFP-op, TDH3pro-yEGFP is replaced by PYK1pro-tGFP and PYK1pro-NES-tGFP (shown in Figure 5A). (C) Principles for determining plasmid copy number and growth rate (fitness) in each condition. The intracellular copy number of the plasmid used in this study varies according to the principle of genetic tug-of-war (gTOW). The copy number of the plasmid is related to the growth rate of the cell. The original genetic tug-of-war method was published in Moriya et al., 2006, and a detailed explanation of the method is also described in Moriya et al., 2012. In the genetic tug-of-war method, the target gene is cloned on a plasmid carrying two selective markers of nutrient requirement, URA3 and leu2-89, and a 2µ plasmid origin (A and B). The plasmid is introduced into the ura3 leu2-deficient strain and first cultured in –Ura medium. This plasmid will be multicopied due to the function of the 2µ origin, and the number of copies in the population will vary. In this case, a vector without an insert (A) results in a copy number of about 25 (C, in Vector/WT). If an excess of the target gene adversely affects growth, the copy number of the plasmid will be lower than the vector even under –Ura conditions, but it will have little effect on growth. For example, the copy number has been found to be about 10 in pTOW40836-TDH3pro-GFP (B), which was used in this study (Eguchi et al., 2018). If we transfer the plasmid-bearing strains to the -Leu/Ura conditions, the copy number of the plasmid rises to about 120. This is because the other marker, leu2-89, is a LEU2 allele with a large deletion in its promoter and is expressed at a significantly lower level than the wild-type LEU2 allele. As a result, cells with high plasmid copy numbers are selected, and the number of copies of the plasmid retained by the population is around 120 copies in the vector case (C, in Vector/WT). In the case of a plasmid incorporating TDH3pro-GFP as a target, the copy number of TDH3pro-GFP also increases with the increase in plasmid copy number caused by leu2-89, resulting in a growth inhibition effect due to GFP overexpression. This acts as a bias to lower the copy number. Thus, the copy number of intracellular plasmids in –Leu/Ura is determined by the tug-of-war between the copy number elevation bias of leu2-89 and the copy number lowering bias of TDH3pro-GFP. In –Leu/Ura conditions, pTOW40836-TDH3pro-GFP has a copy number of about 30, at which time GFP is about 15% of the total protein in the cell (C, in GFP-op/WT) (Eguchi et al., 2018). The relationship between plasmid copy number and growth rate (fitness) can be explained by the figure in (C) When the vector is introduced into the wild type and grown in –Leu/Ura medium, the plasmid copy number rises following the fitness line made by leu2-89. Shown as blue and orange areas are hypothetical histograms of cell populations with plasmids of the copy number shown. The growth rate of cells carrying pTOW40836-TDH3pro-GFP and the copy number of the plasmid depends on the intersection of the fitness line of leu2-89 and the fitness line created by TDH3pro-GFP. This is because this intersection is the highest fitness point for cells cultured with –Leu/Ura. In mutants susceptible to GFP overexpression, the TDH3pro-GFP fitness line shifts to the left (C, in GFP-op/mutant (GFP-op_negative mutants)). This causes the fitness (and plasmid copy number) of this mutant strain overexpressing GFP to be lower than the wild type. In contrast, in mutants resistant to GFP overexpression, the TDH3pro-GFP fitness line shifts to the right (C, in GFP-op/mutant (GFP-op_positive mutants)). This increases the fitness (and plasmid copy number) of this GFP-overexpressing mutant strain over the wild type.