(263b) Stopping Escape and Malfunction of Cells with an Engineered Genetic Code

Abstract:

Engineering a cell for controlled growth and function while enabling new-to-nature biochemistries is invaluable to develop living technologies. Cells with an engineered genetic code are promising biochemical platforms for safe use since they are designed to prevent translation of horizontally transferred DNA. However, we observed that a 61-codon E. coli strain, which lacks TCG/A serine codons (referred together as TCR) and their cognate tRNAs from the genome, can express some genes with TCR codons and yield an escape rate that is orders of magnitude higher than expected. We identified a two-step mechanism of escape in the 61-codon cell through multi-omics data (including DNA sequencing, RNA-seq, and proteomics) and protein language models. To stop escape and malfunction of recoded cells, we engineered kill switches and non-standard amino acid incorporation machineries. We present the first recoded bacterium that can incorporate up to three non-standard amino acids while remaining tightly biocontained. The discovered mechanism of escape provides insights on the flexibility of the genetic code and is a head start toward the controlled use of cells that can incorporate new-to-nature biochemical designs.

During my postgraduate studies, I engineered strains of Escherichia coli for controlled proliferation, function, and use of new biochemistries. Currently, bacteria used as platform technologies rely on a wild-type genetic code, which can result in horizontal gene transfer, escape, and loss of control of the designed programs. Genetic code engineering emerges as a promising alternative since it removes a set of codons and tRNAs from the genome. Without the possibility to read all wild-type codons, bacteria with an engineered genetic code should not translate incoming DNA. Surprisingly, I discovered a new mechanism of escape in bacteria with an engineered genetic code and characterized it with multi-omics data and protein language models. This finding allowed me to develop a biocontainment mechanism that also prevents malfunction of recoded cells. My engineered cell is the first organism that enables the production of proteins containing up to three non-standard amino acids while remaining tightly biocontained and bioisolated.