Quantifying Plasmid Copy Number to Investigate Plasmid Dosage Effects Associated with Directed Protein Evolution

Our laboratory specializes in directed protein evolution, i.e., evolution of proteins under defined selective pressures in the laboratory. Our target genes are encoded in ColE1 plasmids to facilitate the generation of libraries in vivo . We have observed that when random mutations are not restricted to the coding sequence of the target genes, directed evolution results in a strong positive selection of plasmid origin of replication (ori ) mutations. Surprisingly, this is true even during evolution of new biochemical activities, when the activity that is being selected was not originally present. The selected plasmid ori mutations are diverse and produce a range of plasmid copy numbers, suggesting a complex interplay between ori and coding mutations rather than a simple enhancement of level of expression of the target gene. Thus, plasmid dosage may contribute significantly to evolution by fine-tuning levels of activity. Here, we present examples illustrating these observations as well as our methods for efficient quantification of plasmid copy number.