Verification of QS component orthogonality. (A) Genetic schematic of signal orthogonal strains 708 and 709. (B) A 1:1 coculture of 708 and 709 was subjected to the full range of HSLs, and the AUC of the dose–response was compared to predictions based on their original characterization in and . For all predictions, it was assumed the canonical HSL would give the maximum response, so it was assumed providing both HSLs would still give an AUC of 100. Each experiment is normalized by the canonical response, such that the AUC of the GFP dose–response to C6-HSL is set to 100, making the ratio between columns in a set the point of comparison. For example, the 708/709 GFP column set follows this formula: (AUC-C6/AUC- C6), (AUC-pC/AUC-C6), (AUC-Both/AUC-C6). (C, D) Raw fluorescence expression of the 708/709 coculture. Error bars represent SEM (n = 4). (E) Genetic schematic of promoter orthogonal strains 718, 719, and 720. (F) Normalized dose–response AUC showing similarity between predictions and in vivo results. (G, H) Raw fluorescence expression of 720. Raw expression of 718 and 719 is available in . (I) Genetic schematic of complete orthogonal strains 716, 717, and 702. (J) Normalized dose–response AUC of predictions and in vivo results. (K, L) Raw fluorescence expression of 702. Raw expression of 716 and 717 is available in . Error bars represent SEM (n = 4).