Whole-genome evolution of bacteriophage T7 with an expanded genetic code

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Michael Hammerling, Jared W. Ellefson, Andrew D. Ellington, Jeffrey E. Barrick

The University of Texas at Austin, United States

Systems for the in vivo incorporation of unnatural amino acids (UAAs) via amber stop codon suppression have made it possible to expand the genetic codes of many organisms with novel chemical functionalities. However, the long-term effects of access to an artificial genetic code on the global evolution of an organism’s coding sequences remain largely unexplored. Whole-organism experiments of this kind could lead to a better understanding of natural processes of codon reassignment and to more efficient synthetic systems for genome-wide UAA incorporation. We are exploring the consequences of expanded genetic codes through experimental evolution projects involving whole organisms. After serial transfer of T7 bacteriophage on amber-suppressor RF-zero Escherichia coli hosts, we observed mutations leading to UAA incorporation in essential proteins, substitutions that restore stop codons in amber-terminated genes, and addiction such that T7 growth requires an amber-suppressor host. These results pave the way for future experiments that couple the evolution of more efficient UAA incorporation to the survival of addicted organisms and for systematically examining whether genetic codes with certain chemical functionalities lead to greater overall evolvability.