Loss-of-function mutations and evolutionary stability dynamics in re-engineered I7101 circuits with a kanamycin resistance gene. (A) I7101 re-engineering with the addition of a kanamycin resistance (kanR) gene. First the R0010 promoter was added instead of R0011 (top). Then, this circuit was re-engineered to polycistronically transcribe gfp and kanR separately into separate GFP and KanR proteins (middle) and to express a GFP-KanR fusion protein (bottom). (B) Top panel shows the evolutionary stability dynamics of R0010 + E0240 kanR polycistronic circuits propagated with kanamycin (solid green circles) and without kanamycin (open green circles). Bottom panel shows the evolutionary stability dynamics of R0010 + E0240 kanR fusion circuits propagated with kanamycin (solid blue circles) and without kanamycin (open blue circles). R0010 + E0240 and R0011 + E0240 evolutionary stability dynamics are shown in Figure 4. Error bars represent one standard deviation from the mean of nine independently evolved populations. (C) Types of mutations in nine independently evolved populations. For nine independently evolved populations, colored boxes correspond to the mutation legend below the table. The most common mutation for a particular type of mutation is labeled with "1" in the boxes above and less common mutations are labeled with increasing numbers. (D) Most common loss-of-function mutations that inactivated the re-engineered I7101 circuits with a kanamycin resistance gene. See Additional File 1, Supplementary Table S1 for mutation details.