Loss-of-function mutations and evolutionary stability dynamics in I7101. (A) The I7101 genetic circuit. Symbols depict promoters (bent arrows), ribosome binding sites (ovals), coding sequences (arrows), and transcriptional terminators (octagons). I7101 consists of the promoter R0011 and the GFP-expressing element E0240 that is made up of the B0032 RBS, E0040 GFP coding sequence, and B0010-B0012 (B0015) transcriptional terminator. Since lacI is constitutively expressed from the chromosome, it represses GFP expression from the lacI-regulated promoter R0011. When the inducer Isopropyl-beta-D-thiogalactopyranoside (IPTG) is added to the media, it inhibits LacI and activates GFP expression. If no IPTG is in the media, the circuit is off. (B) Evolutionary stability dynamics of R0011 + E0240 evolved under low (-IPTG) and high (+IPTG) 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 IPTG 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 operators within R0011 after 90 generations in the high input evolved populations.