Extended Data Fig. 2: Example conjugation assembly of recoded regions.

Diagrammatic sample of CAGE-mediated assembly of partially recoded ∆TGA regions within rEc∆2E.∆A(EF) & (D). Selectable markers are depicted by rectangles (brown = hyg^R; green = tolC; blue = spec^R; yellow = gent^R; orange = tetA). RK24 plasmid and an oriT-kan^R (Origin of Transfer (oriT) adjacent to a kanamycin selectable marker (kan^R) – depicted by black triangle) are present in all donor strains. Point of oriT-kan^R triangle conveys counter-clockwise directional orientation. Selectable marker (e.g. tolC) is placed 3–5 kb downstream of the oriT. Two selectable markers (Supplementary Table 10) are placed flanking recoded regions to select for and preserve these regions within final strains. To select for multiple recoded regions with greater than two antibiotics, subsequent rounds of selection with additional antibiotics are performed. Marker gent^R was overwritten by conjugal transfer and homologous recombination, while kan^R is excluded by its position upstream of oriT. Offspring strain is confirmed via MASC-PCR of known recoded sites, followed by whole genome sequencing. Graphs and table to the right depict comparative kinetic growth curve data (OD₆₀₀ over time in hours) in nutrient rich (LB) and minimal media (M9) for donor (cyan), recipient (light green), and offspring (dark green) strains. Grey lines correspond to alternative offspring strains resulting from the same conjugation experiment, revealing a diversity of phenotypic results. Non-selected strains displayed poor growth or undesired genotypes (deleterious background mutations or unrecoded TGA codons). Graph displays raw plate reader OD600 readings and DT calculations (n = 1) per strain: parental strains (D & EF) and offspring (DEF & samples 1–6). Figure data are actual and representative of typical experimental results.