Chiappino-Pepe, A.; Radford, F.; Budnik, B.; Tas, H.; Augustin, T. L.; Burgess, H. M.; Moret, M.; Dharani, A. M.; Zheng, Q.; Fan, W.; Afrikawala, M. M.; Thapa, S.; Kuru, E.; Narasimhan, K.; Marchand, J. A.; Perrotta, R. M.; Stokes, J. M.; Lunshof, J. E.; Aach, J. D.; Tam, J. M.; Church, G. M.

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.