Evolutionary Stability of a Refactored Phage Genome

[Image: see text] Engineered genetic systems are commonly unstable; if propagated, they evolve to reverse or modify engineered elements because the elements impair fitness. A goal of synthetic biology is thus to anticipate and avoid detrimental engineering, but little is yet known about which types of elements cause problems in different contexts. In prior work, 30% of the genome of bacteriophage T7 was “refactored” by the insertion or modification of 65 short sequences that included a useful restriction enzyme site in order to, among other goals, separate genes and their translational initiation regions from each other and from other genetic elements. Although gene sequences and known important regions of regulatory elements were kept intact, the translational efficiency of some genes or element regulatory function might have been compromised. We adapted the refactored phage for rapid growth in two conditions, observing fitness and sequence evolution. As anticipated from the original work, refactoring had major fitness effects in both environments, but most of the fitness costs were recovered upon adaptation. The evolved phages retained 60–70% of the design elements, suggesting they had only minor fitness effects. Approximately half the elements that were lost lie within large deletions commonly observed during adaptation of the wild-type genome. Some elements were lost or modified in parallel between the adaptations without affecting T7 gene sequences, but no obvious correlates can be made. Nevertheless, experimental adaptations are useful for identifying specific synthetic design problems, and we suggest that experimental evolution in conjunction with alternative engineering may also be useful in overcoming those problems.