Identifying mutations that enhance the evolutionary stability of fluorescent protein expression from a plasmid in Escherichia coli

Synthetic biologists and metabolic engineers seek to design and create organisms with novel functions. A major difficulty with many designed genetic devices is that they lack evolutionary robustness. In this study, our aim was to identify mutations that could enhance the evolutionary stability of green fluorescent protein (GFP) expression from a plasmid in Escherichia coli. To achieve this goal, we created a mutagenized strain library and performed an evolution experiment. To enrich potential mutants with improved GFP stability, we periodically sorted for cells that remained highly fluorescent as this population was propagated for several hundred generations and less-robust strains accumulated inactivating mutations. Further testing of clones isolated from the final evolved population showed that GFP expression was more stable in these strains and suggested mutations in the chromosome were responsible. Re-sequencing the genomes of four of these strains found that, among other genetic differences from the ancestor, all had a mutation in either PolA or PolB. These two types of DNA polymerase mutations may enhance GFP stability by causing a lower point mutation rate in the E. coli host.