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Genetic Code Expansion refers to an organism containing artificially expanded translational components and modified genetic code in which one or more specific codons have been allocated to encode an amino acid that is not among the 20 "standard" amino acids.
"Standard" or "natural" amino acids are the 20 proteinogenic alpha-amino acids directly encoded by the genetic code in nature and are the building-blocks of all proteins within prokaryotes and eukaryotes. All others are known as "non-standard", "non-canonical", “non-proteinogenic” , or "unnatural". In genetic code expansion many new chemical structures have been engineered into proteins. We use the term “non-canonical amino acid” (ncAA) because it is the most clear and correct for the majority of examples reported.
In the Unnatural Protein Facility we focus on making available genetic code expansion technology that overproduces ncAA-protein in E. coli but in some cases can be moved to other organisms for later study. The UP Facility genetic code expansion methods use orthogonal aminoacyl-tRNA synthetase/tRNACUA pairs that enable the site-specific incorporation of ncAA into proteins in response to the amber stop codon.
The key aspects and function of Genetic Code Expansion have been discussed and reviewed in many publications (see Useful links and resource page). A limited version will be discussed here focusing UP Facility attributes.
Three Stages of Expanded Genetic Code Research and Use
Stage 1 Constructing the orthogonal genetic components (see below) to incorporated the ncAA needed by your research, assembling in the necessary research organism, and checking that the ncAA-protein is generated.
Stage 2 Characterizing and optimizing your ncAA expression system. Many variables alter the ncAA incorporation efficiency, fidelity and permissivity of the ncAA expression system. Sufficient characterization needs to be completed to trust that your ncAA-protein will be produced as expected under the expression conditions used. Efficiency is the measure of full length protein ncAA-protein produced compared to stop codon truncated protein under the expression conditions. Fidelity is the measure of the ratio of ncAA-protein produced compared to any other structure incorporated (natural amino acid or non-standard from the cell). Permissivity is the measure of how many other ncAAs structure can be incorporated by your new ncAA expression system.
Stage 3 Producing your ncAA-protein of interest and studying the structure, interactions, function of the ncAA-protein in vitro or in vivo.
Most researchers are most interested in Stage Three of this process and can be overwhelmed or uninterested in mastering Stages One and Two. The Unnatural Protein Facility is here to help.
Components needed for Genetic Code Expansion
(1) Amino acid that is not toxic to the cell, gets into the cell, and not recognized by natural 20 aminoacyl-tRNA synthetase/tRNA pairs (RS/tRNA).
(2) Engineer an orthogonal aminoacyl synthetase /tRNA pair: meaning this pair works well together, not with organism’s 20 natural pairs, but with organism’s ribosome.
(3) Obtain a codon that is recognized by this new tRNA and no other (in this work a TAG stop or amber codon).
(4) Alter the orthogonal RS active site so it can utilize the new amino acid as a substrate for loading on its tRNA, but not any of the natural 20 amino acids or natural tRNAs.