(225h) Harnessing Orthogonal tRNA for De Novo Generation of Genetic Codes

In living organisms, ribosomal translation transforms genetic information embedded in DNA into actuating components, namely proteins. Though life itself is incredibly diverse at the macroscopic level, at the molecular level, all of life uses the same set of 20 standard amino acid building blocks (with minor exceptions), transfer RNAs (tRNA), and ribosomes to carry out translation. The convergence and association of these interdependent biomolecules is neatly captured in a table known as the ‘standard genetic code’. Even after 3.5 billion years of genetic drift, the ‘standard genetic code’ has been largely refractory to change. In an alternative evolutionary trajectory for life on Earth, would life have settled on using a different set of chemical building blocks and biomacromolecules? In this project, we want to demonstrate that the fundamental language of biological life (namely the ‘standard genetic code’) is not immutable, and can be rewritten inside living cells. If successful, this technology will be a step towards creation of semi- synthetic life and fundamentally change how we use organisms in biomanufacturing.

Humanity has managed to leverage the extant ‘standard genetic code’, containing only 20 amino acids, into multi-billion dollar industries. Biomanufactured proteins are used in medicine as therapeutics (e.g. antibodies, insulin), in industrial processes (e.g. production of high fructose corn syrup), and even in the household (e.g. additives to laundry detergents). These examples highlight the extent of functional utility generated from just 20 amino acid building blocks. If we wanted to start mass producing proteins composed of an alternative set of building blocks, we would be faced by the obstacle posed previously: life has evolved and been optimized around using a very specific set of biomolecules and building blocks.

In this talk, I will be discussing strategies, methods, and results for building a parallel genetic code in living organisms. This talk will focus on the development of a generalizable strategy capable of producing >20 mutually orthogonal transfer RNA (o-tRNA). I will be covering challenges and solutions for producing stable o-tRNAs in cells, specifically in E. coli. While o-tRNAs can form the basis for a parallel genetic code, we are faced with the challenge of building a new genetic code from the ground-up. I will then shift focus cover strategies for populating the new codon table in the absence of a functional orthogonal translation system. If successful, orthogonal genetic codes (as described) have the capacity to translate up to 32 additional amino acids or non-standard building blocks. ( Fields area: chemical biology and synthetic biology)