All existing biological control methods in the context of biomanufacturing–using cells to produce materials–are limited by the genetic information available within the system. Engineering cells with new traits and capabilities requires physically adding and integrating those genetic elements into the system, through molecular cloning, involving DNA assembly, transformation and validation- all complex and time-consuming tasks.
Transfer RNA (tRNA) is found across all domains of life and exists as the bridge between genetic information stored in messenger RNA and the building blocks of proteins, amino acids. In this project, we aim to create a proof of concept for technologies that may use engineered tRNA to 'write' protein sequences using recoded genomes and optogenetics. For the proof-of-concept, we will use engineered tRNAs to change a single codon in a cell's RNA transcript, ultimately altering the protein produced. Specifically, we will use superfolder green fluorescent protein (sfGFP), where alteration of a single amino acid in the protein's chromophore- the part of the molecule responsible for its fluorescence- can shift its normally green fluorescence to blue1. Specifically, our reporter system will contain sfGFP with an amber stop codon (TAG) at position sixty-six.
The second component in our system consists of engineered tRNAs with anticodons that recognise the amber stop codon, causing stop codon suppression at these sites. These suppressor tRNAs can ‘override’ a premature stop signal in an mRNA transcript, causing the ribosome to read through the stop codon and incorporate the amino acid charged to the tRNA. We have engineered two suppressor tRNAs that cause incorporation of tyrosine or histidine at amber stop codons, producing full-length green or blue fluorescent proteins, respectively. We have deployed these suppressor tRNAs into a recoded E. coli strain (c321ΔA)2, in which all amber stop codons in coding sequences have been replaced, enabling efficient amber suppression by our engineered tRNA. Lastly, we will outline a plan to light-activate these suppressor tRNAs using photo-switchable antisense blocking oligonucleotides.
In this research, we hope to establish the first instance of light-activated tRNAs for real-time manipulation of protein sequences. Rather than switching the expression of entire genes on or off, as is common in Synthetic Biology, our approach would enable precise spatiotemporal control of translation at the codon level through optogenetic regulation of tRNAs. By directly controlling amino acid incorporation during translation, this strategy could reduce reliance on iterative molecular cloning. Such capabilities could be transformative for on-demand biomanufacturing in resource-limited environments.