Visualization of Evolutionary Stability Dynamics and Competitive Fitness of Escherichia coli Engineered with Randomized Multigene Circuits

Strain engineering for synthetic biology and metabolic engineering applications often requires the expression of foreign proteins that can reduce cellular fitness. In order to quantify and visualize the evolutionary stability dynamics in engineered populations of Escherichia coli , we constructed randomized CMY (cyan-magenta-yellow) genetic circuits with independently randomized promoters, ribosome binding sites, and transcriptional terminators that express cyan fluorescent protein (CFP), red fluorescent protein (RFP), and yellow fluorescent protein (YFP). Using a CMY color system allows for a spectrum of different colors to be produced under UV light since the relative ratio of fluorescent proteins vary between circuits, and this system can be used for the visualization of evolutionary stability dynamics. Evolutionary stability results from 192 evolved populations (24 CMY circuits with 8 replicates each) indicate that both the number of repeated sequences and overall expression levels contribute to circuit stability. The most evolutionarily robust circuit has no repeated parts, lower expression levels, and is about 3-fold more stable relative to a rationally designed circuit. Visualization results show that evolutionary dynamics are highly stochastic between replicate evolved populations and color changes over evolutionary time are consistent with quantitative data. We also measured the competitive fitness of different mutants in an evolved population and find that fitness is highest in mutants that express a lower number of genes (0 and 1 > 2 > 3). In addition, we find that individual circuits with expression levels below 10% of the maximum have significantly higher evolutionary stability, suggesting there may be a hypothetical "fitness threshold" that can be used for robust circuit design.