Tuning evolvability via plasmid copy number and regulatory architecture

Genetic modules are often designed and implemented with inspiration from engineering disciplines. Although this approach can be successful because of the similarities underpinning physical and biochemical systems, it neglects a key factor that affects the performance of living organisms: evolution. Thus, it is crucial to incorporate the impact of inevitable mutations into the design and analysis of genetic modules. Combining computational modeling and in vivo mutagenesis experiments in Escherichia coli, we characterize how the interplay of gene dosage via plasmid copy number (PCN) and regulatory architecture affect the phenotypic mutation rate. For example, while greater PCN facilitates the emergence of gain-of-function mutations, it instead curbs the spread of loss-of-function mutations. We further reveal that mutations in the coding region are often masked at the phenotypic level, unlike those occurring in the regulatory region which become more prominent as PCN increases, both when the regulator is expressed constitutively and when it is self-repressed. Together, our results shed light on evolutionary organizing principles and aid the rational design of both evolutionarily stable and highly evolvable biocircuits.