Fig. 5: Escapee analysis of SPC110-ERdd and engineering for improved containment stringency.

a Growth of SPC110-ERdd at different estradiol concentrations in YPD. b SPC110-ERdd containment escape mutations. Eight SPC110-ERdd escapees were sequenced, and seven escape mutations were found. All resulted in a premature translation stop by mutation to a stop codon or a frameshift, mapping to the C-terminal region of SPC110 or the linker, and conversely removal of the ERdd tag. No mutations were found in ERdd itself. The dispensable C-terminal region of SPC110 in which escape mutations clustered is marked with a red dashed line. c Growth of SPC110Δ845-ERdd, in which the C-terminal region has been removed, at different estradiol concentrations in YPD. As with SPC110-ERdd, growth is fully restored by 100 nM estradiol. Growth assays were performed with three biological replicates. Source data for a and c are provided as a Source Data file. d SPC110Δ845-ERdd containment stringency is markedly improved over SPC110-ERdd. 23 SPC110Δ845-ERdd escapees were sequenced. 14 separate escape mutations were found, with all but one of the sampled SPC110Δ845-ERdd escape mutations mapping to the linker or the N-terminal region of the ERdd tag. Residues are numbered separately for the SPC110 coding sequence, the 10 amino acid linker (shown in gray), and the ERdd tag.