AGENTEX automates the testing of new protein assembly rules in a mixture of disrupted cell components
AGENTEX automates the testing of new protein assembly rules in a mixture of disrupted cell components
On August 26, the authors of a Nature paper described AGENTEX, an automated system for constructing and testing alternative genetic codes in lysate, a mixture of components from disrupted Escherichia coli cells. In one configuration, 22 tRNAs, the molecules that carry amino acids, defined a code that left 14 codons available for new assignments.
The genetic code maps three-letter RNA sequences, called codons, to amino acids. The ribosome reads these triplets and joins the corresponding amino acids into a protein. The standard code contains 64 codons: 61 encode amino acids, three act as stop signals, and several different triplets often encode the same amino acid. A codon that has been freed from its original role can be assigned to another amino acid.
An engineered tRNA can already change a single rule. In experiments with a suppressor tRNA, the tRNA read a premature UGA stop codon as arginine, allowing the cell to complete the protein. AGENTEX extends this approach by testing entire sets of rules. Such experiments require the new code to operate separately from the standard protein assembly system.
Reassigning a codon in a living bacterium first requires replacing every instance of its original use in the genome. In a 2013 study by George Church's group, researchers replaced all 321 occurrences of one codon in the Escherichia coli genome and then reassigned that codon to another amino acid. AGENTEX performs an earlier stage of testing in lysate. For each variant, researchers can assemble a set of rules, produce a peptide, and verify its composition.
The authors began with tRNAs. Most of these molecules end in the sequence CCA. A method called tSCAN tested which artificially assembled tRNAs the lysate could charge with an amino acid. Many variants with modified ends worked, particularly those containing the sequence CGA. Standard ribosomes retained their usual assembly rules. After two point substitutions in ribosomal RNA, however, the ribosomes accepted CGA-tRNAs and assembled peptides according to the new code. This allows the standard and experimental sets of rules to operate in parallel within the same cell-free mixture.
The automated procedure prepares a tRNA pool, an RNA template, and the reaction, while the measurement guides the selection of the next code variant. In one experiment, the authors exchanged the assignments of serine and alanine and assigned the UAG stop codon to para-azidophenylalanine, a synthetic building block. In lysate containing modified ribosomes, the light signal indicating assembly of the complete test peptide was approximately six times higher than in the same experiment with standard ribosomes. Mass spectrometry confirmed the specified substitutions in the peptides.
The number “34” describes the capacity of the scheme: 20 standard amino acids and 14 codons available for new building blocks. In the paper, the authors demonstrated a 22-tRNA code containing one such amino acid. AGENTEX turns the design of each subsequent variant into a reproducible laboratory cycle: specify the tRNA composition, produce a peptide, verify its composition, and revise the rules for the next reaction.