Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Engineered Overlapping Genes Paired with Directed Evolution Prolongs the Evolutionary Stability of a Genetic Circuit


Jennifer Chlebek, Christina Kang-Yun, Sean Leonard, Dante P. Ricci, Mimi Yung, Yongqin Jiao, and Dan Park* (


Lawrence Livermore National Laboratory



A primary goal of this Science Focus Area (SFA) project is to establish genetic sequence entanglement as a generalizable biocontainment strategy to protect engineered functions against mutational inactivation and to mitigate the horizontal transfer of invasive genes. Sequence entanglement was inspired by overlapping genes found in many viral genomes and involves the synthetic encoding (entangling) of two genes within the same DNA sequence space through use of alternative reading frames. As such, mutations within the entangled region likely impact the function of both genes, providing a mechanism to constrain the allowable mutational space. The team thus hypothesize that by entangling a gene-of-interest (GOI) with an essential gene, the evolution of the GOI can be constrained by rendering mutations in one frame non-permissible due to deleterious mutations in the frame encoding the essential gene. As a proof-of-concept, researchers assessed the utility of an entanglement design in which a toxin is entangled with an essential gene, to improve genetic stability of a kill-switch circuit.


The development of synthetic biological circuits that maintain functionality over application relevant timescales remain a significant challenge. Synthetic circuits are often burdensome to cellular fitness and are subject to evolutionary pressures, which select for mutated and non-functional circuits. In this study, researchers employed a gene overlap technique called synthetic sequence entanglement—in which one protein is encoded entirely within an alternate reading frame of another gene—to enhance the sequence stability of a burdensome engineered genetic circuit. Specifically, the toxin-encoding relE gene was entangled within ilvA, which encodes threonine deaminase, an enzyme essential for isoleucine biosynthesis. This pairing allows the ability to test whether an essential function (isoleucine biosynthesis) can increase the mutational robustness of a gene prone to mutational inactivation (e.g., relE).

Starting from a partially functional entanglement design in which significant missense mutations (~79% of entangled residues) were introduced within the ilvA sequence to accommodate a wild-type amino acid sequence for RelE, the team made targeted modifications of an internal ribosome binding site that simultaneously enhanced the expression of the RelE toxin and the function of IlvA. Using this optimized design, researchers show that entanglement of relE with ilvA significantly increased the evolutionary stability of the toxic relE gene, which retained function for >130 generations. This stabilizing effect was achieved through a complete alteration of the allowable mutational landscape such that mutations inactivating both entangled gene products were disfavored. Instead, small deletions, insertions, and point mutations accumulated within the regulatory region of ilvA for the majority of lineages. By reducing baseline relE expression, these more benign mutations lowered circuit burden, which suppressed the accumulation of relE inactivating mutations, thereby prolonging kill-switch function. Overall, this work demonstrates the utility of sequence entanglement to increase the evolutionary stability of burdensome synthetic circuits.

Funding Information

This work is supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Lawrence Livermore National Laboratory Secure Biosystems Design SFA “From Sequence to Cell to Population: Secure and Robust Biosystems Design for Environmental Microorganisms”. Work at LLNL is performed under the auspices of the U.S. Department of Energy at Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344 (LLNL-ABS-845282).