Adaptation of Bacteriophage on Synthetic Transcription Machinery

With the emergence of synthetic biology, it remains unclear if synthetic parts (i.e. altered activity, substrate specificity, orthogonality, etc.) can significantly alter the evolutionary trajectory of the host organism. In this study, T7 bacteriophages lacking their wild-type RNA polymerase (T7Δ1) were passaged on an engineered T7 RNA polymerase designed to recognize an orthogonal promoter. T7 RNA polymerase plays a key step in the life cycle to the bacteriophage as it controls expression of 17 critical promoters. We hypothesize that massive changes in the regulatory architecture would emerge to better accommodate the orthogonal RNA polymerase. After 100 passages on the orthogonal polymerase, we observed multiple changes in multiple promoters and the evolved strains had a higher overall fitness than the ancestor, reaching that of wild-type T7 bacteriophage. Additionally, the evolved strains were able to accommodate insertion of the orthogonal RNA polymerase back into their genomes, whereas the ancestor T7Δ1 was not. This provides an example of how simple design strategies may frequently require further evolutionary optimization. Our work raises the question whether synthetic biology can be used for laboratory speciation events and the development of orthogonal viruses.