Loss-of-function mutations and evolutionary stability dynamics in T9002. (A) The T9002 genetic circuit. Symbols depict promoters (bent arrows), ribosome binding sites (ovals), coding sequences (arrows), and transcriptional terminators (octagons). T9002 consists of two devices, a luxR receiver device and a GFP-expressing device. The first device is composed of the tetR-regulated promoter R0040 that is constitutively expressed in the MG1655 strain since it does not produce TetR, B0034 RBS, C0062 luxR coding sequence, and B0010-B0012 (B0015) transcriptional terminator. The second device is composed of the R0062 luxR promoter, B0032 RBS, E0040 GFP coding sequence, and B0015 transcriptional terminator. LuxR is constitutively expressed from the tetR promoter. When the inducer 3OC6HSL (AHL) is added to the media, it binds with LuxR to activate transcription of GFP from the luxR promoter. If no AHL is in the media, the circuit is off. (B) Evolutionary stability dynamics of T9002 evolved under low (-AHL) and high (+AHL) input conditions. Low and high input evolved populations are shown with solid gray triangles and solid black circles, respectively. Evolved populations at different timepoints were grown with AHL to measure relative GFP levels. Relative fluorescence normalized by OD is plotted vs. generations. Error bars represent one standard deviation from the mean of nine independently evolved populations. (C) This circuit repeatedly has a deletion between homologous repeated terminators after 30 generations in the high input evolved populations.