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

Principles Governing Expression of Overlapping Genes


Bentley Lim* (, Sean P. Leonard, Tiffany M. Halvorsen, Dante Ricci, Mimi C. Yung, Yongqin Jiao


Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory


A primary goal of the Lawrence Livermore National Laboratory BioSecure Science Focus Area (SFA) is to establish gene overlaps—in which two genes are encoded within the same DNA sequence through use of alternative reading frames—as a generalizable biocontainment strategy to protect engineered functions against mutational inactivation and to mitigate the horizontal transfer of invasive genes. This project is focused on determining the biophysical and molecular principles governing expression of overlapping genes. The project seeks to (1) identify the biophysical mechanisms and constraints underlying expression of overlapping genes and (2) predict and engineer future overlapping gene used in microbes for deployment.


In synthetic biology, methods for stabilizing genetically engineered functions in intended hosts are necessary to cope with mutation accumulation. One generalizable strategy to preserve genetic information is through gene overlaps, translating two distinct proteins from the same mRNA in different open reading frames. Overlapping a sequence with an essential gene can alter its fitness landscape and produce a constrained evolutionary path. While ongoing work is focused on large-scale redesign of the entangled proteins to satisfy sequence constraints required by sequence overlap, little is known about how expression is affected for entangled genes and its ramifications on the success of developing successful gene entanglements. To dissect the role of entanglement on gene expression, researchers have devised a methodological pipeline of genetic entanglement by inserting a protein encoded in an alternate reading frame with an external gene, minimizing amino acid changes to both genes, permitting functionality of both overlapped genes. Researchers demonstrate the creation and evaluation of multiple overlapping, out-of-frame insertion designs in flexible loops of inner membrane anchored antibiotic resistance alleles encoding efflux pumps. The team shows that inserted genes (toxins and fluorescent reporters) can function despite their location inside another coding sequence in an alternate frame and that function of both genes can be retained with minimal redesign. Interestingly, the team finds that the nested genes exhibit significant variability in the expression based on the location of the insertion of the external gene. This variability is not due to differences in mRNA levels but manifests at the level of protein abundance. To further generate a broad understanding of expression alterations during gene-nesting, researchers have performed insertional profiling to generate libraries of additional external genes (encoding for cytoplasmic globular proteins) with a nested gfp gene located throughout the sequence of the external gene. After identifying nested entanglements with full functionality of the external gene, the team will perform a series of mechanistic studies (Structure-Seq and Ribo-Seq) to identify whether and how expression may be altered for both entangled genes. These studies will provide general principles that underlie the expression of engineered entangled and nested genes with the goal of creating entangled genes capable of expressing at levels needed to stabilize function of both genes. Ultimately, this work will establish general guidelines for designing gene entanglements for improved stability of engineered genetics and circuits in microbes deployed in situ.

Funding Information

This work is supported by the DOE, Office of Science, BER program, Lawrence Livermore National Laboratory (LLNL) BioSecure Science Focus Area within the Secure Biosystems Design program. Work at LLNL is performed under the auspices of the DOE at LLNL under contract no. DE-AC52-07NA27344.