Genetic Code Expansion
The genetic code for all life is based upon four nucleotides, 64 codons, and 20 amino acids. Yet in the past two decades, biologists have expanded the genetic code by redirecting specific codons to encode amino acids beyond the 20 standard amino acids.
Expanding the Genetic Code
In protein translation, an aminoacyl-tRNA synthetase (aaRS) loads its cognate tRNA with a specific amino acid. Then, the tRNA is pulled into the ribosome and if the anticodon on the tRNA can bind to the mRNA (hence, the anticodon is complementary to the codon), the amino acid from the tRNA is incorporated into the growing peptide chain.
To expand the genetic code, modified tRNAs, codons, and tRNA synthetases are introduced into the cell on plasmids and the new amino acid is introduced in the media. Generally, you will need two plasmids, as depicted in the figure below:
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A plasmid expressing the tRNA and its cognate aminoacyl-tRNA-synthetase (aaRS) that has been evolved to incorporate non-canonical amino acids (ncAAs).
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A plasmid containing the gene of interest with the modified codon (typically the amber codon) that is recognized by the cognate charged tRNA.
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Once these plasmids have been introduced in the cells, the non-canonical amino acid can be incorporated using the existing protein translation machinery.

To expand the genetic code, 4 major changes to the standard translation machinery are needed in order to incorporate a non-canonical amino acid into the protein of interest:
- The non-canonical amino acid, which is generally introduced in the media.
- A new codon to be allocated to the new amino acid. Because there are no free codons, this can be challenging. In E.coli, the rarest codon is the amber stop codon (UAG) and thus this codon is often used. The gene of interest can be expressed from a plasmid containing a UAG codon at the place where the new amino acid would be incorporated. Other options, such a 4-base pair codons, have also been utilized.
- A tRNA that recognizes this codon.
- An aminoacyl-tRNA synthetase to load the new amino acid onto the tRNA. The tRNA and synthetase are called an orthogonal set, because they should not crosstalk with the endogenous tRNA and synthetase sets. Many of these sets are derived from M. jannaschii, M. barkeri, or E.coli and can be mutated and screened through directed evolution to charge the tRNA with a different amino acid. They are typically expressed from a single plasmid, with multiple copies of the tRNA.
Applications
By making small changes in selected amino acids within a protein, any alterations in structure or function in the protein can be observed. The introduced amino acid can also be used to intentionally change the activity of a protein (e.g. converting a DNA binding protein to a DNA cleaving enzyme) or to regulate the activity of a protein so that it is responsive to specific stimuli, such as light. On a broader scale, the expanded genetic code can help us understand and evolve proteins for various purposes from therapeutics to biopolymers.
Tips for Success
Before beginning to reprogram the genetic code, there are several things to consider. If you are working off of a previously established protocol, make sure to match the growth medium, ncAA concentration, and the cell lines used previously.
Remember that the orthogonal pairs of synthetase and tRNA that work for one organism may not work for another. The orthogonal synthetase must aminoacylate only the orthogonal tRNA, and not endogenous ones. Endogenous synthetases cannot aminoacylate the orthogonal tRNA. And the orthogonal tRNA has to bind to an unallocated codon. Therefore many controls must be used to make sure that these conditions are true. Always express first with a control reporter gene – GFP for E. coli or mCherry-GFP for mammalian cells. You should also express the protein with and without the ncAA in the media to make sure that the full length protein is only made when the ncAA is included.
Browse Synthetase Plasmids
The table below highlights plasmids that contain aminoacyl tRNA synthetase for use in E.coli and Mammalian Cells. Many of the plasmids also contain one or more copies of the cognate tRNA gene.
ID | Plasmid | Synthetase | Origin | ncAA | Organism | Codon | PI | |
---|---|---|---|---|---|---|---|---|
31186 | pEVOL-pAzF | p-azidohenylalanine RS | Methanocaldococcus janaschii | p-azido-l-phenylalanine | E. coli | TAG | Peter Schultz | |
31190 | pEVOL-pBpF | p-benzoylphenylalanine RS | Methanocaldococcus janaschii | p-benzoyl-l-phenylalanine | E. coli | TAG | Peter Schultz | |
48215 | pULTRA-CNF | tyrosyl synthetase | Methanocaldococcus janaschii | para-cyanophenylalanine (pCNPhe) | E. coli | TAG | Peter Schultz | |
48696 | pANAP | AnapRS | E. coli | fluorescent AA, Anap | Mammalian | TAG | Peter Schultz | |
49086 | pDULE-ABK | pyrrolysyl tRNA sythetase | Methanosarcina barkeri | aliphatic diazirine amino acid | E. coli and Mammalian cells | TAG | Peter Schultz | |
50831 | pAcBac2.tR4-OMeYRS/GFP* | tyrosyl-tRNA synthetase | E. coli | various unnatural amino acids | Mammalian | TAG | Peter Schultz | |
50832 | pAcBac1.tR4-MbPyl | pyrrolysyl-tRNA synthetase | Methanosarcina barkeri | variety of unnatural amino acids | Mammalian | TAG | Peter Schultz | |
51401 | pAM1 | NLL-MetRS | E. coli | azi-donorleucine (Anl) | Y. enterocolitica | ATG | David Tirrell | |
62598 | pKPY514 | phenylalanyl-tRNA synthetase subunit | E. coli | p-azido-L-phenylalanine (Azf) | C. elegans | ATG | David Tirrell | |
62599 | pKPY197 | phenylalanyl-tRNA synthetase (CePheRS) | C.elegans | p-azido-L-phenylalanine (Azf) | C. elegans | ATG | David Tirrell | |
63177 | pMarsL274G | methionyl-tRNA synthetase (L274GMmMetRS) | murine mutant | azidonorleucine (Anl) | Mammalian | ATG | David Tirrell | |
64915 | pMAH-POLY | tyrosyl synthetase | E. coli | pBof | Mammalian | TAG | Huiwang Ai | |
68292 | SepOTSλ | SepRS9 | E. coli | Sep | E. coli | TAG | Jesse Rinehart | |
71403 | pCMV-DnpK | pyrrolysyl-tRNA-synthetase | Methanosarcina barkeri | N6‐(2‐(2,4‐dinitrophenyl)acetyl)lysine (DnpK) | E. coli and Mammalian | TAG | Huiwang Ai | |
71404 | pMAH2-CageCys | leucyl-tRNA-synthetase | E. coli | photocaged cysteine | Mammalian | TAG | Huiwang Ai | |
73544 | pEvol-pAcFRS.2.t1 | pAcFRS.2.t1 | E. coli | p-acetyl-l-phenylalanine (pAcF) | E. coli | TAG | Farren Isaacs | |
73545 | pEvol-pAcFRS.1.t1 | pAcFRS.1.t1 | E. coli | p-acetyl-l-phenylalanine (pAcF) | E. coli | TAG | Farren Isaacs | |
73546 | pEvol-pAzFRS.2.t1 | pAzFRS.2.t1 | E. coli | p-azido-l-phenylalanine (pAzF) | E. coli | TAG | Farren Isaacs | |
73547 | pEvol-pAzFRS.1.t1 | pAzFRS.1.t1 | E. coli | p-azido-l-phenylalanine (pAzF) | E. coli | TAG | Farren Isaacs | |
82417 | pUltra-sY | sY-specific aaRS | Methanocaldococcus janaschii | sulfotyrosine (sY) | E. coli and rice | TAG | Chang Liu | |
85484 | pDule-tfmF A65V S158A | tri-fluoromethyl-phenylalanine synthetase | Methanocaldococcus janaschii | family of [19]F-UAAs | E. coli | TAG | Ryan Mehl | |
85494 | pDule-pCNF | para-cyanophenylalanine synthetase | Methanocaldococcus janaschii | azidoPhenylalanine | E. coli | TAG | Ryan Mehl | |
85495 | pDule2-pCNF | para-cyanophenylalanine synthetase | Methanocaldococcus janaschii | azidoPhenylalanine | E. coli | TAG | Ryan Mehl | |
85496 | pDule-Tet2.0 | Tetrazine2.0 tRNA synthetase | Methanocaldococcus janaschii | Tetrazine 2.0 | E. coli | TAG | Ryan Mehl | |
85497 | pDule2-Tet2.0 | Tetrazine2.0 tRNA synthetas | Methanocaldococcus janaschii | Tetrazine 2.0 | E. coli | TAG | Ryan Mehl | |
85498 | pDule-3-nitroTyrosine (5B) | 3NY (5B) synthetase | Methanocaldococcus janaschii | 3-nitroTyrosine | E. coli | TAG | Ryan Mehl | |
85499 | pDule2-3-nitroTyrosine (5B) | 3NY (5B) synthetase | Methanocaldococcus janaschii | 3-nitroTyrosine | E. coli | TAG | Ryan Mehl | |
85500 | pDule-IBBN (G2) | IBBN (G2) synthetase | Methanocaldococcus janaschii | 4-(2′-bromoisobutyramido)-phenylalanine (IBBN) and structurally analogous amino acids | E. coli | TAG | Ryan Mehl | |
85501 | pDule2-IBBN (G2) | IBBN (G2) synthetase | Methanocaldococcus janaschii | 4-(2′-bromoisobutyramido)-phenylalanine (IBBN) and structurally analogous amino acids | E. coli | TAG | Ryan Mehl | |
85502 | pDule-para-aminoPhe | pAF synthetase | Methanocaldococcus janaschii | para-aminoPhe | E. coli | TAG | Ryan Mehl | |
85503 | pDule2-para-aminoPhe | pAF synthetase | Methanocaldococcus janaschii | para-aminoPhe | E. coli | TAG | Ryan Mehl | |
89189 | pMaRSC | methionyl-tRNA synthetase (L274GMmMetRS) | murine mutant | azidonorleucine (Anl) | Mammalian | ATG | David Tirrell | |
91705 | pSupAR-MbPylRS(DiZPK) | pyrrolysyl-tRNA synthetase | Methanosarcina barkeri | photocrosslinkers DiZPK, DiZSeK, or DiZHSeC | E. coli | TAG | Peng Chen | |
91706 | pCMV-MbPylRS(DiZPK) | pyrrolysyl-tRNA synthetase | Methanosarcina barkeri | photocrosslinkers DiZPK, DiZSeK, or DiZHSeC | Mammalian | TAG | Peng Chen | |
92047 | pCOTS-pyl-GFP(35TAG) | Pyrrolysyl tRNA synthetase | Methanosarcina mazei | cyanobacterial | TAG | Lital Alfonta | ||
92048 | gCOTS-pyl | Pyrrolysyl tRNA synthetase | Methanosarcina mazei | cyanobacterial | TAG | Lital Alfonta | ||
99222 | pTECH-chPylRS(IPYE) | Pyrrolysyl tRNA synthetase | Chimeric | p-iodo-L-phenylalanine | E. coli | TAG | Dieter Soll | |
104069 | pTECH-chAcK3RS(IPYE) | AcK3RS | Chimeric | Nε-acetyl-L-lysine | E. coli | TAG | David Liu | |
104070 | pTECH-MbAcK3RS(IPYE) | AcK3RS | Methanosarcina barkeri | Nε-acetyl-L-lysine | E. coli | TAG | David Liu | |
104071 | pTECH-MmAcK3RS(IPYE) | AcK3RS | Methanosarcina mazei | Nε-acetyl-L-lysine | E. coli | TAG | David Liu | |
104072 | pTECH-MbPylRS(IPYE) | Pyrrolysyl tRNA synthetase | Methanosarcina barkeri | m-iodo-L-phenylalanine | E. coli | TAG | David Liu | |
104073 | pTECH-MmPylRS(IPYE) | Pyrrolysyl tRNA synthetase | Methanosarcina mazei | m-iodo-L-phenylalanine | E. coli | TAG | David Liu | |
105829 | pIRE4-Azi | Azi-tRNA synthetase (EAziRS) | humanized | p-Azido-phenylalanine (Azi) | Mammalian | TAG | Irene Coin | |
105830 | pNEU-hMbPylRS-4xU6M15 | Pyrrolysyl tRNA synthetase | Methanosarcina barkeri | Pyl-like click amino acids, tRNA M15 | Mammalian | TAG | Irene Coin | |
113644 | pRF0G-Tyr | tyrosine tRNA synthetase | Methanococcus jannaschii | tyrosine | E. coli | TAG | Jeffrey Barrick | |
113645 | pRF0G-IodoY | iodotyrosine tRNA synthetase | Methanococcus jannaschii | iodotyrosine | E. coli | TAG | Jeffrey Barrick | |
122650 | Mm-PylRS-AF/Pyl-tRNACUA | Pyrrolysyl tRNA synthetase | Methanosarcina mazei | trans-cyclooct- 2-ene-lysine (TCOK) | Mammalian | TAG | Howard Hang | |
126035 | pEVOL-ABK | pyrrolysyl tRNA sythetase | Methanosarcina barkeri | 3’-azibutyl-N-carbamoyl-lysine (AbK) | E. coli | TAG | Andrea Musacchio | |
127411 | pEVOL-pylT-N346A/C348A | pyrrolysyl tRNA sythetase | Methanosarcina mazei | mono-substituted phenylalanine derivatives and tyrosinyl ethers | E. coli | TAG | Wenshe Liu | |
137908 | PylRS-AS | pyrrolysyl tRNA sythetase | Methanosarcina mazei | fluorophenylalanine derivatives | E. coli | TAG | Wenshe Liu | |
137976 | pEVOL-AckRS | pyrrolysyl tRNA sythetase | Methanosarcina mazei | AzHeK | E. coli | TAG | Wenshe Liu | |
140009 | pAS_4xMma PylT_FLAG-Mma PylRS | pyrrolysyl tRNA sythetase | Methanosarcina mazei | CpK, AbK | Mammalian | TAG | Simon Elsaesser | |
140011 | pAS_4xMx PylT_FLAG-Mx PylRS | pyrrolysyl tRNA sythetase | Methanomethylophilus alvus Mx1201 | CpK | Mammalian | TAG | Simon Elsaesser | |
140012 | pAS_4xMx PylT_FLAG-Mx PylRS YA | Simon Elsaesser | ||||||
140013 | pAS_4xMx PylT C41CA_FLAG_Mx PylRS | pyrrolysyl tRNA sythetase | Methanomethylophilus alvus Mx1201 | Mammalian | TAG | Simon Elsaesser | ||
140014 | pAS_4xMx PylT C41CA_FLAG_Mx PylRS YA | pyrrolysyl tRNA sythetase | Methanomethylophilus alvus Mx1201 | Mammalian | TAG | Simon Elsaesser | ||
140018 | pAS_4xBstTyrT(CUA)_EcoTyrRS-FLAG | tyrosine tRNA synthetase | E. coli | Mammalian | TAG | Simon Elsaesser | ||
140019 | pAS_4xEcoLeuT(CUA)_AnapRS | AnapRS | E. coli | azido-phenylalanine | Mammalian | TAG | Simon Elsaesser | |
140020 | pAS_4xMma PylT_FLAG-AbKRS-chIPYE | photo-crosslinking-lysine | Simon Elsaesser | |||||
140021 | pAS_4xMma PylT_FLAG-AcKRS | AcKRS | Methanosarcina mazei | acetyl-lysine | Mammalian | TAG | Simon Elsaesser | |
140022 | pAS_4xMma PylT_FLAG-PcKRS | PcKRS | Methanosarcina barkeri | photocaged-lysine | Mammalian | TAG | Simon Elsaesser | |
140023 | pAS_4xMma PylT_FLAG-Mma PylRS AF | Simon Elsaesser | ||||||
141173 | pAcBac1-3nitroY-A7-RS | A7 3nY tRNA synthetase | Methanosarcina barkeri | Ryan Mehl | ||||
141174 | pAcBac1-haloTyrRS C6 | C6 HaloTyr tRNA synthetase | Ryan Mehl | |||||
153557 | pEvol-MjaYRS | MJaYRS | Methanococcus jannaschii | 3-aminotyrosine | E.coli | TAG | Huiwang Ai | |
153558 | pMAH-EcaYRS | EcaYRS | E. coli | 3-aminotyrosine | Mammalian | Huiwang Ai | ||
154762 | pAS_FLAG-Mma PylRS | Methanosarcina mazei | Simon Elsaesser | |||||
154763 | pAS_FLAG-G1 PylRS | G1 PylRS | methanogenic archaeon mixed culture ISO4-G1 | Simon Elsaesser | ||||
154764 | pAS_FLAG-Mx1201 PylRS | PylRS | Methanomethylophilus alvus Mx1201 | Simon Elsaesser | ||||
154768 | pAS_4xG1 PylT FLAG-G1 PylRS | G1 PylRS | methanogenic archaeon mixed culture ISO4-G1 | Simon Elsaesser | ||||
154769 | pAS_4xG1 PylT FLAG-G1 PylRS Y125A | G1 PylRS Y125A | methanogenic archaeon mixed culture ISO4-G1 | Simon Elsaesser | ||||
154773 | pAS_4xhybPylT A41AA C55A FLAG-G1 PylRS Y125A | G1 PylRS Y125A | methanogenic archaeon mixed culture ISO4-G1 | Simon Elsaesser | ||||
154774 | pAS_4xU6-PylT M15 (UUA) FLAG-Mma PylRS | PylRS | Methanosarcina mazei | ochre | Simon Elsaesser | |||
157925 | pMAH-POLY-eRF1(E55D) | POLY-eRF1(E55D) | E. coli | para-substituted phenylalanine analogs | Mammalian | TAG | Huiwang Ai | |
160377 | pDule-Mb haloTyrRS C6 | C6 HaloTyrosine tRNA synthatase | Methanosarcina barkeri | E. coli | Ryan Mehl | |||
160378 | pDule2-Mb haloTyrRS C6 | C6 HaloTyrosine tRNA synthatase | Methanosarcina barkeri | E. coli | Ryan Mehl |
Browse Strains
The table below highlights bacteria strains that have been modified to enhance non-standard amino acid incorporation.
ID | Strain | Description | PI | |
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48999 | C321 | all TAG sites changes to UAG | Church | |
48998 | C321.ΔA | all TAG sites changes to UAG, RF1 function removed | Church | |
49018 | C321.ΔA.exp | all TAG sites changes to UAG, RF1 function removed, MutS restored so decreased mutation rate | Church | |
98564 | C321.Ub-UAG-sfGFP | all TAG sites changes to UAG, RF1 function removed, with Ubiquitin-UAG-sfGFP reporter | Church | |
98565 | C321.ΔClpS.Ub-UAG-sfGFP | all TAG sites changes to UAG, RF1 function removed, ClpS inactivated, with Ubiquitin-UAG-sfGFP reporter | Church | |
68306 | C321.ΔA | all TAG sites changes to UAG, RF1 function removed, deletion of SerB to maintain sufficient levels of Sep in the cytoplasm for protein synthesis | Rinehart | |
73581 | C321.deltaA (Isaacs lab) | all TAG sites changes to UAG, RF1 function removed | Isaacs | |
87359 | C321.∆A.opt | all TAG sites changes to UAG, RF1 function removed, improved doubling time. | Church | |
69493 | MCJ.559 | TAG sites, RF1 function removed, genomic deletions for improved incorporation | Jewett | |
69495 | rEc.13.delA | 13 amber sites changed to TAA, RF1 function removed | Jewett |
Browse Target Plasmids
The table below highlights plasmids that contain genes with modified codons for unnatural amino acid incorporation.
ID | Plasmid | Gene | Expression Type | PI | |
---|---|---|---|---|---|
105666 | pBad-CA TAG20 | carbonic anhydrase II | Bacterial Expression | Mehl | |
105667 | pBad-CA TAG93 | carbonic anhydrase II | Bacterial Expression | Mehl | |
105668 | pBad-CAts TAG97 | carbonic anhydrase II | Bacterial Expression | Mehl | |
105836 | pBad-CA TAG126 | carbonic anhydrase II | Bacterial Expression | Mehl | |
105837 | pBad-CA TAG186 | carbonic anhydrase II | Bacterial Expression | Mehl | |
105838 | pBad-CA TAG233 | carbonic anhydrase II | Bacterial Expression | Mehl | |
105839 | pBad-HPII | hydroperoxidase II catalase | Bacterial Expression | Mehl | |
105843 | pBad-HPII TAG283 | hydroperoxidase II catalase | Bacterial Expression | Mehl | |
105844 | pBad-HPII TAG348 | hydroperoxidase II catalase | Bacterial Expression | Mehl | |
105845 | pBad-HPII TAG568 | hydroperoxidase II catalase | Bacterial Expression | Mehl | |
105846 | pBad-HPII TAG206 | hydroperoxidase II catalase | Bacterial Expression | Mehl | |
105847 | pBad-HPII TAG415 | hydroperoxidase II catalase | Bacterial Expression | Mehl | |
105848 | pBad-HPII TAG392 | hydroperoxidase II catalase | Bacterial Expression | Mehl | |
85483 | pBad-sfGFP 150TAG | sfGFP 150TAG | Bacterial Expression | Mehl | |
85482 | pBad-sfGFP | sfGFP | Bacterial Expression | Mehl | |
82501 | pGLO-GFP-3UAG | GFP-3UAG | Bacterial Expression | Liu | |
82500 | pGLO-GFP-1UAG | GFP-1UAG | Bacterial Expression | Liu | |
64913 | pcDNA3-pnGFP | pnGFP | Mammalian Expression | Ai | |
122475 | HA-IFITM3-F8-TAG | IFITM3 | Mammalian Expression | Hang | |
122476 | HA-IFITM3-F8-TAG-C71A | IFITM3-C71A | Mammalian Expression | Hang | |
122477 | HA-IFITM3-F8-TAG-C72A | IFITM3-C72A | Mammalian Expression | Hang | |
122478 | HA-IFITM3-F8-TAG-C105A | IFITM3-C105A | Mammalian Expression | Hang | |
122479 | HA-IFITM3-V93-TAG | IFITM3 | Mammalian Expression | Hang | |
122480 | IFITM3-N21del-V93-TAG | IFITM3-N21del | Mammalian Expression | Hang | |
140016 | pAS_4xMx PylT_GFP 150TAG | sfGFP 150 TAG stop | Mammalian Expression | Elsaesser | |
140008 | pAS | empty backbone | Mammalian Expression | Elsaesser |