Abstract
Engineering the genetic code restricts DNA transfer (cellular bioisolation) and enables new chemistries via non-standard amino acid incorporation. These distinct properties make recoded cells state-of-the-art safe technologies. However, evolutionary pressures may endanger the longevity of the recoding. Here, we reveal that recoded Escherichia coli lacking 18,214 serine codons and two tRNASer can express wild-type antibiotic resistance genes and escape up to seven orders of magnitude faster than expected. We show a two-step escape process whereby recoded cells mistranslate antibiotic resistance genes to survive until modified or mutated tRNAs reintroduce serine into unassigned codons. We developed genetic-code-sensitive kill switches that sense serine incorporation and prevent cellular escape while preserving encoding of three distinct non-standard amino acids. This work lays the foundation for the long-term controlled function of cells that incorporate new chemistries, with implications for the design, use, and biosafety of synthetic genomes in clinical and environmental applications where physical containment is insufficient.
Competing Interest Statement
A.C-P., F.R., and G.M.C. are listed as inventors of a provisional patent application filed through Harvard Medical School. Q.Z. and W.F. are employees at GenScript U.S.A. Inc.. GenScript U.S.A. Inc., Q.Z., and W.F. synthesized all new plasmids and did not design or execute experiments for this project. G.M.C. is a founder of companies with related financial interests: GRO Biosciences, EnEvolv (Ginkgo Bioworks), Pearl Bio. Other relevant financial interests of G.M.C. are listed at http://arep.med.harvard.edu/gmc/tech.html.
