Creating overlapping genes by alternate-frame insertion

Overlapping genes-wherein two different proteins are translated from alternative frames of the same DNA sequence-provide a means to stabilize an engineered gene by directly linking its evolutionary fate with that of an overlapped gene. However, creating overlapping gene pairs is challenging as it requires redesign of both protein products to accommodate overlap constraints. Here, we present a new "overlapping, alternate-frame insertion" (OAFI) method for creating overlapping genes by insertion of an "inner" gene, encoded in an alternate frame, into a flexible region of an "outer" gene. Using OAFI, we create new overlapping gene pairs of bacterial toxins within an antibiotic resistance gene. We show that both the inner and outer genes retain functionality despite redesign, with translation of the inner gene is influenced by its overlap position in the outer gene. Additionally, we show that selection for the outer gene alters the permitted inactivating mutations in the inner gene and that overlapping toxins can restrict horizontal gene transfer of the antibiotic resistance gene. Overall, OAFI offers a versatile tool for synthetic biology, expanding the applications of overlapping genes in gene stabilization and biocontainment.