Expanding the functions of a 57 codon recoded E.coli genome

A fundamental and highly conserved code is embedded in all genomes and underlies biological life. This code is known as the ‘standard genetic code’, which defines how genetic information translates amino acid building blocks into proteins during their biological manufacturing process. Harnessing the universality of the standard genetic code has revolutionized the material, food, chemical, and energy industry.

Although minor deviations from the standard code exist in a few organisms, the standard genetic code has withstood nearly 3.5 billion years of genetic drift by being highly conserved across all domains of life. The genetic codon is a triplet unit of a genetic material (DNA or RNA) that can define a protein building block. The mapping of 64 triplet codons to the 20 canonical amino acids, the building blocks of proteins, results in the same amino acid being encoded by multiple synonymous codons.

By rationally building synthetic genomes in living cells, with a reduced set of synonymous codons, the liberated codons can be reassigned to incorporate non-natural amino acids into proteins. By systematic codon replacement, researchers are progressing towards the final assembly of a computationally designed bacterial genome that relies on 57 codons instead of the universal 64 codons.

In this project, the fully assembled 57-codon synthetic bacteria cell will be explored and further modified for safe bio-containment and genetic isolation to prevent exchange of genetic information with the environment. The researchers will also build an open-access database to enable streamlined computational approaches for non-canonical amino acid incorporation.

As a form of outreach, the researchers will organize a two-day open webinar series annually with philosophers, early-career researchers, STEM graduate students, and scientific experts engaged in synthetic cell engineering projects globally to discuss future perspectives and ethical issues associated with synthetic cells.

The standard genetic code maps the 64 triplet codons in the genome with the 20 canonical amino acids. Translating the information encoded in the standard genetic code to proteins further involves adaptor tRNA molecules, ribosomes, and amino-acyl tRNA synthetases. Researchers are progressing towards assembling a 57-codon Escherichia coli genome by replacing seven codons that encode the canonical amino acids serine, leucine, alanine and the amber stop codon with their synonymous alternatives.

The liberated codons will incorporate multiple non-canonical amino acids and establish biocontainment for engineered genetic information. The researchers will gain fundamental insights on cellular plasticity by studying the adaptations of the proteome towards long-term non-canonical amino acid dependence. The study will also provide biological insights into genetic code evolution by tracking for processes such as codon-capture and ambiguous decoding during the long-term adaptation and dependence of the recoded genome to non-canonical amino acids.

The project will also build an open-access database for genetic code expansion and make available computational tools to facilitate the research with non-canonical amino acids. In sum, it is expected that this project will provide new tools and knowledge for academic and industrial efforts related to genetic code and synthetic cell engineering while simultaneously facilitating the safe use of synthetic biological systems and communicating these aspects to a wide range of researchers and STEM students.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.