Fitness landscape of substrate-adaptive mutations in evolved APC transporters

Growth rate calculations:

Single colonies of S. cerevisiae Δ10AA pADHXC3GH-GOI were inoculated in YB media supplemented with 4 mm NH₄⁺ and 0.1 mg/ml ampicillin, and grown until late logarithmic phase. The cultures were pelleted at 750 × g for 10 min at 30 °C and washed with YB media. The wells in the microplate were filled with the amino acids of interest to a final concentration of 2 mM and with culture cells to a final OD₆₀₀ of 0.04, to a final total well volume of 200 µl. Sterile water was added in the space between the wells to avoid evaporation. The prepared microplates included three biological replicates of the strains with the plasmid containing the gene of interest (GOI) and one biological replicate of the strain with the empty vector. The absorbance in each well was measured at 600 nm in 30 min intervals without shaking of the microplate, at 30 °C for 72 h in a SpectraMax ABS Plus plate reader. The data sets (CSV files) are the raw optical density readings from the growth assays, along with plate layout metadata (CSV files). The growth rates were derived based on the Baranyi growth model, using the growthrates package in R.

Included transporter genes:

Mutated transporters (script growth_rates_mutants_Baranyi2.r)

AGP1, AGP1-N, AGP1-V, AGP1-NV, AGP1-G, AGP1-T, PUT4, PUT4-S

Wild-type transporters (script growth_rates_wild_types_Baranyi2.r)

AGP1, BAP2, CAN1, HIP1, LYP1, MMP1, PUT4

Plasmid sequences (Genbank files):

pADHXC3GH-AGP1, pADHXC3GH-BAP2, pADHXC3GH-CAN1, pADHXC3GH-HIP1, pADHXC3GH-LYP1, pADHXC3GH-MMP1, pADHXC3GH-PUT4-S L207S, pADHXC3GH-PUT4, pADHXC3GH

Measureing relative membrane fluorescence (all transporters in this study have C-terminal GFP tags):

Micrographs were analyzed with ImageJ using the script analyse_cell_perimeter.ijm