Fig. 5: Fitness-oriented screening method to identify permissive residues for OMeY substitution at the N-terminus of Cdc4 and Cdc27 proteins.
a Schematic diagram showing the workflow of the fitness-oriented screening method. To replace the codon corresponding to individual residues at the N-terminus (from residue 4 to 20) of CDC4 and CDC27 genes with TAG, synthesized DNA libraries were transformed and integrated onto the genome of the transcriptional safeguard strain (PGAL1-RPS3) via URA3 marker-assisted homologous recombination. Then, desired two-layered safeguard strains showing robust growth could be identified based on the growth curve analysis from all obtained OMeY auxotrophs. Doubling time of two-layered safeguard strains derived from Cdc4- (b) and Cdc27-based (c) OMeY auxotrophs respectively. The blue dotted line represents the doubling time of the control strains (PGAL1-RPS3 strain containing the URA3 marker integrated at the upstream of wild-type CDC4 and CDC27 genes respectively). The error bars show the mean and SD of three biological replicates (*p < 1 × 10−2; **p < 1 × 10−4). p values were determined by two-sided, unpaired Student’s t test. The exact p value is provided in Source Data file. The x-axis shows different residues that are tolerant for OMeY substitution; four residues were labeled in red to represent corresponding safeguard strains that were confirmed to have undetectable escape when monitored for 14 days (assay limit is ~1.1 and ~1.2 × 10−9 escapees per CFU for (b) and (c), respectively). Source data are provided as a Source Data file.
