Engineering gene overlaps to sustain genetic constructs in vivo

Evolution is often an obstacle to the engineering of stable biological systems due to the selection of mutations inactivating costly gene circuits. Gene overlaps induce important constraints on sequences and their evolution. We show that these constraints can be harnessed to increase the stability of costly genes by purging loss-of-function mutations. We combine computational and synthetic biology approaches to rationally design an overlapping reading frame expressing an essential gene within an existing gene to protect. Our algorithm succeeded in creating overlapping reading frames in 80% of E. coli genes. Experimentally, scoring mutations in both genes of such overlapping construct, we found that a significant fraction of mutations impacting the gene to protect have a deleterious effect on the essential gene. Such an overlap thus protects a costly gene from removal by natural selection by associating the benefit of this removal with a larger or even lethal cost. In our synthetic constructs, the overlap converts many of the possible mutants into evolutionary dead-ends, reducing the evolutionary potential of the system and thus increasing its stability over time.