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 | Expression | Codon | PI | |
|---|---|---|---|---|---|---|---|---|
| 31186 | pEVOL-pAzF | p-azidohenylalanine RS | M. jannaschii | p-azido-l-phenylalanine | Bacterial | TAG | Peter Schultz | |
| 31190 | pEVOL-pBpF | p-benzoylphenylalanine RS | M. jannaschii | p-benzoyl-l-phenylalanine | Bacterial | TAG | Peter Schultz | |
| 48215 | pULTRA-CNF | tyrosyl-tRNA synthetase | M. jannaschii | para-cyanophenylalanine (pCNPhe) | Bacterial | TAG | Peter Schultz | |
| 48696 | pANAP | AnapRS | E. coli | fluorescent AA, Anap | Mammalian | TAG | Peter Schultz | |
| 49086 | pDULE-ABK | PylRS | M. barkeri | aliphatic diazirine amino acid | Bacerial, Mammalian | 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 | PylRS | M. barkeri | variety of unnatural amino acids | Mammalian | TAG | Peter Schultz | |
| 51401 | pAM1 | NLL-MetRS | E. coli | azi-donorleucine (Anl) | Bacterial | 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) | M. musculus | azidonorleucine (Anl) | Mammalian | ATG | David Tirrell | |
| 64915 | pMAH-POLY | tyrosyl-tRNA synthetase | E. coli | pBof | Mammalian | TAG | Huiwang Ai | |
| 68292 | SepOTSλ | SepRS9 | E. coli | Sep | Bacterial | TAG | Jesse Rinehart | |
| 71403 | pCMV-DnpK | PylRS | M. barkeri | N6‐(2‐(2,4‐dinitrophenyl)acetyl)lysine (DnpK) | Bacterial, 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) | Bacterial | TAG | Farren Isaacs | |
| 73545 | pEvol-pAcFRS.1.t1 | pAcFRS.1.t1 | E. coli | p-acetyl-l-phenylalanine (pAcF) | Bacterial | TAG | Farren Isaacs | |
| 73546 | pEvol-pAzFRS.2.t1 | pAzFRS.2.t1 | E. coli | p-azido-l-phenylalanine (pAzF) | Bacterial | TAG | Farren Isaacs | |
| 73547 | pEvol-pAzFRS.1.t1 | pAzFRS.1.t1 | E. coli | p-azido-l-phenylalanine (pAzF) | Bacterial | TAG | Farren Isaacs | |
| 82417 | pUltra-sY | sY-specific aaRS | M. jannaschii | sulfotyrosine (sY) | Bacterial, Plant | TAG | Chang Liu | |
| 85484 | pDule-tfmF A65V S158A | tri-fluoromethyl-phenylalanine synthetase | M. jannaschii | family of [19]F-UAAs | Bacterial | TAG | Ryan Mehl | |
| 85494 | pDule-pCNF | para-cyanophenylalanine synthetase | M. jannaschii | azidoPhenylalanine | Bacterial | TAG | Ryan Mehl | |
| 85495 | pDule2-pCNF | para-cyanophenylalanine synthetase | M. jannaschii | azidoPhenylalanine | Bacterial | TAG | Ryan Mehl | |
| 85496 | pDule-Tet2.0 | Tetrazine2.0 tRNA synthetase | M. jannaschii | Tetrazine 2.0 | Bacterial | TAG | Ryan Mehl | |
| 85497 | pDule2-Tet2.0 | Tetrazine2.0 tRNA synthetas | M. jannaschii | Tetrazine 2.0 | Bacterial | TAG | Ryan Mehl | |
| 85498 | pDule-3-nitroTyrosine (5B) | 3NY (5B) synthetase | M. jannaschii | 3-nitroTyrosine | Bacterial | TAG | Ryan Mehl | |
| 85499 | pDule2-3-nitroTyrosine (5B) | 3NY (5B) synthetase | M. jannaschii | 3-nitroTyrosine | Bacterial | TAG | Ryan Mehl | |
| 85500 | pDule-IBBN (G2) | IBBN (G2) synthetase | M. jannaschii | 4-(2′-bromoisobutyramido)-phenylalanine (IBBN) and structurally analogous amino acids | Bacterial | TAG | Ryan Mehl | |
| 85501 | pDule2-IBBN (G2) | IBBN (G2) synthetase | M. jannaschii | 4-(2′-bromoisobutyramido)-phenylalanine (IBBN) and structurally analogous amino acids | Bacterial | TAG | Ryan Mehl | |
| 85502 | pDule-para-aminoPhe | pAF synthetase | M. jannaschii | para-aminoPhe | Bacterial | TAG | Ryan Mehl | |
| 85503 | pDule2-para-aminoPhe | pAF synthetase | M. jannaschii | para-aminoPhe | Bacterial | TAG | Ryan Mehl | |
| 89189 | pMaRSC | methionyl-tRNA synthetase (L274GMmMetRS) | M. musculus | azidonorleucine (Anl) | Mammalian | ATG | David Tirrell | |
| 91705 | pSupAR-MbPylRS(DiZPK) | PylRS | M. barkeri | photocrosslinkers DiZPK, DiZSeK, or DiZHSeC | Bacterial | TAG | P. Chen | |
| 91706 | pCMV-MbPylRS(DiZPK) | PylRS | M. barkeri | photocrosslinkers DiZPK, DiZSeK, or DiZHSeC | Mammalian | TAG | P. Chen | |
| 92047 | pCOTS-pyl-GFP(35TAG) | PylRS | M. mazei | Cyanobacterial | TAG | Lital Alfonta | ||
| 92048 | gCOTS-pyl | PylRS | M. mazei | Cyanobacterial | TAG | Lital Alfonta | ||
| 99222 | pTECH-chPylRS(IPYE) | PylRS | Chimeric | p-iodo-L-phenylalanine | Bacterial | TAG | Dieter Söll | |
| 104069 | pTECH-chAcK3RS(IPYE) | AcK3RS | Chimeric | Nε-acetyl-L-lysine | Bacterial | TAG | David Liu | |
| 104070 | pTECH-MbAcK3RS(IPYE) | AcK3RS | M. barkeri | Nε-acetyl-L-lysine | Bacterial | TAG | David Liu | |
| 104071 | pTECH-MmAcK3RS(IPYE) | AcK3RS | M. mazei | Nε-acetyl-L-lysine | Bacterial | TAG | David Liu | |
| 104072 | pTECH-MbPylRS(IPYE) | PylRS | M. barkeri | m-iodo-L-phenylalanine | Bacterial | TAG | David Liu | |
| 104073 | pTECH-MmPylRS(IPYE) | PylRS | M. mazei | m-iodo-L-phenylalanine | Bacterial | TAG | David Liu | |
| 105829 | pIRE4-Azi | Azi-tRNA synthetase (EAziRS) | p-Azido-phenylalanine (Azi) | Mammalian | TAG | Irene Coin | ||
| 105830 | pNEU-hMbPylRS-4xU6M15 | PylRS | M. barkeri | Pyl-like click amino acids, tRNA M15 | Mammalian | TAG | Irene Coin | |
| 113644 | pRF0G-Tyr | tyrosyl-tRNA synthetase | M. jannaschii | tyrosine | Bacterial | TAG | Jeffrey Barrick | |
| 113645 | pRF0G-IodoY | iodotyrosine tRNA synthetase | M. jannaschii | iodotyrosine | Bacterial | TAG | Jeffrey Barrick | |
| 122650 | Mm-PylRS-AF/Pyl-tRNACUA | PylRS | M. mazei | trans-cyclooct- 2-ene-lysine (TCOK) | Mammalian | TAG | Howard Hang | |
| 126035 | pEVOL-ABK | PylRS | M. barkeri | 3’-azibutyl-N-carbamoyl-lysine (AbK) | Bacterial | TAG | Andrea Musacchio | |
| 127411 | pEVOL-pylT-N346A/C348A | PylRS | M. mazei | mono-substituted phenylalanine derivatives and tyrosinyl ethers | Bacterial | TAG | Wenshe Liu | |
| 127415 | pEVOL-AcKRS-CloDF | PylRS | M. mazei | acyl-lysine derivatives | Bacterial | Wenshe Liu | ||
| 127445 | pEDF-PhdRS | PylRS N346A-C348A | M. mazei | Bacterial | Wenshe Liu | |||
| 137908 | PylRS-AS | PylRS | M. mazei | fluorophenylalanine derivatives | Bacterial | TAG | Wenshe Liu | |
| 137976 | pEVOL-AckRS | PylRS | M. mazei | AzHeK | Bacterial | TAG | Wenshe Liu | |
| 140009 | pAS_4xMma PylT_FLAG-Mma PylRS | PylRS | M. mazei | CpK, AbK | Mammalian | TAG | Simon Elsaesser | |
| 140011 | pAS_4xMx PylT_FLAG-Mx PylRS | PylRS | M. alvus | CpK | Mammalian | TAG | Simon Elsaesser | |
| 140012 | pAS_4xMx PylT_FLAG-Mx PylRS YA | PylRS YA | M. alvus | Mammalian | TAG | Simon Elsaesser | ||
| 140013 | pAS_4xMx PylT C41CA_FLAG_Mx PylRS | PylRS | M. alvus | Mammalian | TAG | Simon Elsaesser | ||
| 140014 | pAS_4xMx PylT C41CA_FLAG_Mx PylRS YA | PylRS | M. alvus | Mammalian | TAG | Simon Elsaesser | ||
| 140018 | pAS_4xBstTyrT(CUA)_EcoTyrRS-FLAG | tyrosyl-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 | chimeric PylRS | Chimeric | photo-crosslinking-lysine | Mammalian | TAG | Simon Elsaesser | |
| 140021 | pAS_4xMma PylT_FLAG-AcKRS | AcKRS | M. mazei | acetyl-lysine | Mammalian | TAG | Simon Elsaesser | |
| 140022 | pAS_4xMma PylT_FLAG-PcKRS | PcKRS | M. barkeri | photocaged-lysine | Mammalian | TAG | Simon Elsaesser | |
| 140023 | pAS_4xMma PylT_FLAG-Mma PylRS AF | PylRS AF | M. mazei | Mammalian | TAG | Simon Elsaesser | ||
| 141173 | pAcBac1-3nitroY-A7-RS | A7 3nY tRNA synthetase | M. barkeri | Mammalian | Ryan Mehl | |||
| 141174 | pAcBac1-haloTyrRS C6 | C6 HaloTyrosine tRNA synthatase | E. coli | Mammalian | Ryan Mehl | |||
| 153557 | pEvol-MjaYRS | MJaYRS | M. jannaschii | 3-aminotyrosine | Bacterial | TAG | Huiwang Ai | |
| 153558 | pMAH-EcaYRS | EcaYRS | E. coli | 3-aminotyrosine | Mammalian | Huiwang Ai | ||
| 154762 | pAS_FLAG-Mma PylRS | PylRS | M. mazei | Mammalian | TAG | Simon Elsaesser | ||
| 154763 | pAS_FLAG-G1 PylRS | G1 PylRS | M. archaeon | Mammalian | TAG | Simon Elsaesser | ||
| 154764 | pAS_FLAG-Mx1201 PylRS | PylRS | M. alvus | Mammalian | TAG | Simon Elsaesser | ||
| 154768 | pAS_4xG1 PylT FLAG-G1 PylRS | G1 PylRS | M. archaeon | Mammalian | TAG | Simon Elsaesser | ||
| 154769 | pAS_4xG1 PylT FLAG-G1 PylRS Y125A | G1 PylRS Y125A | M. archaeon | Mammalian | TAG | Simon Elsaesser | ||
| 154773 | pAS_4xhybPylT A41AA C55A FLAG-G1 PylRS Y125A | G1 PylRS Y125A | M. archaeon | Mammalian | TAG | Simon Elsaesser | ||
| 154774 | pAS_4xU6-PylT M15 (UUA) FLAG-Mma PylRS | PylRS | M. mazei | Mammalian | TAA | Simon Elsaesser | ||
| 157925 | pMAH-POLY-eRF1(E55D) | POLY-eRF1(E55D) | E. coli | para-substituted phenylalanine analogs | Mammalian | TAG | Huiwang Ai | |
| 160041 | pRaGE Pyl TAG GFP Y35TAG | PylRS | M. mazei | Bacterial | TAG | Lital Alfonta | ||
| 160089 | pPaGE Pyl TAG FliC T248TAG | PylRS | M. mazei | Bacterial | TAG | Lital Alfonta | ||
| 160377 | pDule-Mb haloTyrRS C6 | C6 HaloTyrosine tRNA synthatase | M. barkeri | Halotyrosine Amino Acids | Bacterial | TAG | Ryan Mehl | |
| 160378 | pDule2-Mb haloTyrRS C6 | C6 HaloTyrosine tRNA synthatase | M. barkeri | Halotyrosine Amino Acids | Bacterial | TAG | Ryan Mehl | |
| 163915 | pRSF-ChPylTMSK | ChPylTMSK | N6-(((Trimethylsilyl)methoxy)carbonyl)-L-lysine (TMSK) | Bacterial | TAG | Thomas Huber | ||
| 163916 | pXPR-SF61 | SF5Phe tRNA synthetase | para-Pentafluorosulfanyl-Phenylalanine | Bacterial | TAG | Thomas Huber | ||
| 164080 | pCMV-MtPylRS (human-opti) | PylRS | M. thermophila | acetyl-lysine | Mammalian | Tao Liu | ||
| 164081 | pCMV-MfPylRS (human-opti) | PylRS | M. flavescens | acetyl-lysine | Mammalian | Tao Liu | ||
| 164195 | pCMV-MtAcKRS (human-opti) | AcKRS | M. thermophila | acetyl-lysine | Mammalian | Tao Liu | ||
| 164196 | pCMV-MfBulKRS (human-opti) | BulKRS | M. flavescens | acetyl-lysine | Mammalian | Tao Liu | ||
| 164579 | pEVOL-pAzF[TAG] | pCNFRS | M. jannaschii | pAzF, pCNF, or pENF | Bacterial | TAG | Ryan Mehl | |
| 164580 | pUltraI-Tet3.0[TAA] | Tet3.0RS | M. barkeri | Tet3.0 | Bacterial | TAA | Ryan Mehl | |
| 172482 | pEVOL-pylT-AznLRS | AznLRS | M. mazei | azidonorleucine | Bacterial | TAG | Wenshe Liu | |
| 173897 | SepRS(2)/pSertRNA(B4)/EF-Sep | aminoacyl-tRNA synthetase | M. maripaludis | phosphoserine | Bacterial | Jason W Chin | ||
| 174078 | pDule-3-nitroTyrosine (A7) | 3NY (A7) synthetase | M. jannaschii | 3-nitroTyrosine | Bacterial | TAG | Ryan Mehl | |
| 174079 | pDule2-3-nitroTyrosine (A7) | 3NY (A7) synthetase | M. jannaschii | 3-nitroTyrosine | Bacterial | TAG | Ryan Mehl | |
| 174080 | pDule-Tet3.0 | Tet3.0 tRNA synthetase | M. barkeri | Tetrazine 3.0 | Bacterial | TAG | Ryan Mehl | |
| 174081 | pAcBac1-NES-Flag-R2-84-MbRS Tetrazine3.0 | NES-Flag-R2-84 tRNA synthetase | M. barkeri | Tetrazine 3.0 | Mammalian | Ryan Mehl | ||
| 174099 | pDule-Acd82 | Acridone 82 tRNA synthetase | M. barkeri | acridone | Bacterial | TAG | Ryan Mehl | |
| 174100 | pDule2-Acd82 | Acridone 82 tRNA synthetase | M. barkeri | acridone | Bacterial | TAG | Ryan Mehl | |
| 174101 | pAcBac1.tR4-AcdRS82 | Acridone 82 tRNA synthetase | M. barkeri | acridone | Mammalian | Ryan Mehl | ||
| 174515 | pMB1_MmPylRS_MmtRNA-Pyl-opt(UGA) | PylRS | M. mazei | Bacterial | TGA | Jason W Chin | ||
| 174516 | pMB1_1R26PylRS(CbzK)_AfTyrRS(p-I-Phe)_AlvtRNA-ΔNPyl(8)(CGA)_AftRNA-Tyr(A01)(CUA) | 1R26PylRS and AfTyrRS | CbzK and p-I-Phe | Bacterial | TCG and TAG | Jason W Chin | ||
| 174718 | pRSF-G1mCNPRS | G1mCNPRS | M. archaeon | L-3-(2-cyano-4-pyridyl)alanine (mCNP) | Bacterial | TAG | Thomas Huber | |
| 174719 | pRSF-G1pCNPRS | G1pCNPRS | M. archaeon | L-3-(2-cyano-5-pyridyl)alanine (pCNP) | Bacterial | TAG | Thomas Huber | |
| 174890 | pAS_8xMmaPylT_EF1_FLAG-MmaPylRS | PylRS | M. mazei | Mammalian | TAG | Simon Elsaesser | ||
| 174896 | pAS_4xMmaPylT(UUA)_EF1_MmaPylRS | PylRS | M. mazei | Mammalian | TAA | Simon Elsaesser | ||
| 174899 | pAS_4xMmaPylT(UCA)_EF1_MmaPylRS | PylRS | M. mazei | Mammalian | TGA | Simon Elsaesser | ||
| 174901 | pAS_MmaPylT_EF1_NES-MmaPylRS WT | PylRS | M. mazei | Mammalian | TAG | Simon Elsaesser | ||
| 174902 | pAS_MmaPylT_EF1_NES-MmaPylRS AF | PylRS (Y306A, Y384F) | M. mazei | Mammalian | TAG | Simon Elsaesser | ||
| 177310 | pRSF-G1(7FTrp)RS | PylRS | M. archaeon | 7-Fluoro-L-Tryptophan (7FTrp) | Bacterial | TAG | Thomas Huber | |
| 177311 | pRSF-G1TFAKRS | M. archaeon | N6-(trifluoroacetyl)-L-lysine (TFA-Lys) | Bacterial | TAG | Thomas Huber | ||
| 182287 | pcDNA3.1(+)_U6 tRNAPyl_CMV NESPylRS(AF) | Y306A/Y384F (AF) pyrrolysine (Pyl) tRNA synthetase | M. mazei | Mammalian | TAG | Ivana Nikić-Spiegel | ||
| 182537 | pEVOL-MmAcKRS1-PylTUUA | PylRS | M. mazei | Bacterial | TAA | Wenshe Liu | ||
| 182653 | pcDNA3.1(+)_U6 tRNAPyl_CMV NESPylRS(AF)_IRES_eRF1(E55D)-HA | Y306A/Y384F (AF) pyrrolysine (Pyl) tRNA synthetase | M. mazei | Mammalian | TAG | Ivana Nikić-Spiegel | ||
| 182881 | Mut1-RS | tyrosyl-tRNA synthetase | M. jannaschii | 4-propargyloxy-l-phenylalanine (pPR) | Bacterial | TAG | Miriam Amiram | |
| 182882 | Mut2-RS | tyrosyl-tRNA synthetase | M. jannaschii | 4-propargyloxy-l-phenylalanine (pPR) | Bacterial | TAG | Miriam Amiram | |
| 182884 | AzoRS-4 | tyrosyl-tRNA synthetase | M. jannaschii | azobenzene-bearing uAAs | Bacterial | TAG | Miriam Amiram | |
| 188537 | pSerOTS-C1* (V70) | SerRS | M. maripaludis | Phosphoserine | Bacterial | TAG | Jesse Rinehart | |
| 193250 | pMV361_MjTyrRS | tyrosyl-tRNA synthetase | M. jannaschii | Bacterial | TAG | Peter Schultz | ||
| 196486 | pcPylRS(Y384F Y306A) | PylRS(Y384F Y306A) | M. mazei | Z-Lys or Azido-Z-Lys | Mammalian | TAG | Kensaku Sakamoto | |
| 197099 | pIYN3 | TyrRS | M. jannaschii | halogenated tyrosines | Bacterial | TAG | Kensaku Sakamoto | |
| 197100 | pCDF-Mm2 | PylRS | M. mazei | pyrrolysine derivatives | Bacterial | TAG | Kensaku Sakamoto | |
| 197101 | pCDF-Az | TyrRS | M. jannaschii | azidophenylalanine | Bacterial | TAG | Kensaku Sakamoto | |
| 197102 | pBpaRS3tRNA | TyrRS | M. jannaschii | p-benzoylphenylalanine | Bacterial | TAG | Kensaku Sakamoto | |
| 197566 | pAcBac1-Ma-Acridone-RS1 (A7) | PylRS | M. alvus | acridonyl-alanine | Mammalian | TAG | Ryan Mehl | |
| 197573 | pBK- Ma PylRS WT | PylRS | M. alvus | Bacterial | Ryan Mehl | |||
| 197576 | pAcBac1-Mb-Fluoro-Phe B5 | Pyl Fluoro-Phe B5 | M. barkeri | fluorinated phenylalanine derivatives | Mammalian | TAG | Ryan Mehl | |
| 197577 | pAcBac1-Mb-Fluoro-Phe D6 | Pyl Fluoro-Phe D6 | M. barkeri | fluorinated phenylalanine derivatives | Mammalian | TAG | Ryan Mehl | |
| 197651 | pDule- Mj Acd A9 | Acd-A9 | M. jannaschii | acridone | Bacterial | TAG | Ryan Mehl | |
| 197652 | pDule2 - Mj Acd A9 | Acd-A9 | M. jannaschii | acridone | Bacterial | TAG | Ryan Mehl | |
| 197930 | pSUP-(2xCouRS-tRNAopt) | 7-HCou Aminoacyl | (7-hydroxy-4-coumarin-yl) ethylglycine | Bacterial | TAG | Kenneth Johnson | ||
| 198323 | pRSF-G1TMSNKRS | M. archaeon | TMSNK | Bacterial | TAG | Thomas Huber | ||
| 200225 | pMega-MaPylRS | PylRS | M. alvus | Nε-Boc-lysine | Bacterial | TAG | Alanna Schepartz | |
| 200226 | pMega-MaFRSA | PylRS | M. alvus | meta-trifluoromethylphenylalanine | Bacterial | TAG | Alanna Schepartz | |
| 201922 | pERM2-nhpSer | SepRS | M. maripaludis | non-hydrolyzable phosphoserine | Bacterial | TAG | Ryan Mehl | |
| 207516 | pRSF-G1(7AW)RS | G1(7AW)RS | Methanogenic archaeon | 7-Aza-L-Tryptophan (7AW) | Bacterial | TAG | Thomas Huber | |
| 207620 | pRSF-G1(4/5FTrp)RS | G1(4/5FTrp)RS | Methanogenic archaeon | 4-Fluoro-L-Tryptophan (4FTrp), 5-Fluoro-L-Tryptophan (5FTrp) | Bacterial | TAG | Thomas Huber | |
| 207621 | pRSF-G1(6/7FTrp)RS | G1(6/7FTrp)RS | Methanogenic archaeon | 6-Fluoro-L-Tryptophan (6FTrp), 7-Fluoro-L-Tryptophan (7FTrp) | Bacterial | TAG | Thomas Huber | |
| 207639 | pEVOL-NBK-1 | PylRS | M. mazei | Pyrrolysine | Bacterial | Cole DeForest | ||
| 209738 | pAB228i16 | G1 PylRS | Methanogenic archaeon | Bacterial | Ahmed Badran | |||
| 209739 | pAB228i24 | G1 PylRS | Methanogenic archaeon | Bacterial | Ahmed Badran | |||
| 209740 | pAB228q12 | TyrRS | M. jannaschii | Bacterial | Ahmed Badran | |||
| 209741 | pAB228r3 | Mlum1RS | Bacterial | Ahmed Badran | ||||
| 209742 | pAB228p | PylRS | M. mazei | Bacterial | Ahmed Badran | |||
| 209743 | pAB228p4 | PylRS | M. mazei | Bacterial | Ahmed Badran | |||
| 209744 | pAB228v | TrpRS | S. cerevisiae | Bacterial | Ahmed Badran | |||
| 209745 | pAB228v6 | TrpRS | S. cerevisiae | Bacterial | Ahmed Badran | |||
| 209746 | pAMC070n1a12 | ScTrpRS, Lum1PylRS, MmPylRS | S. cerevisiae, Lum1, M. mazei | Bacterial | Ahmed Badran | |||
| 212125 | pBK-Ma pylRS nitroY/haloY-F5 | PylRS | M. alvus | 3-nitrotyrosine (3-nitro-Y), 3-halotyrosine (3-halo-Y) | Bacterial | TAG | Ryan Mehl | |
| 214359 | pAJE3-E7 | aminoacyl-tRNA synthetase | M. jannaschii | 1,2,4,5-tetrazine ncAAs | Bacterial | TAG | Ryan Mehl | |
| 217361 | pIDTSmart-MbPylRS | PylRS | M. barkeri | azidolysine (AzK) | Mammalian | TAG | Abhishek Chatterjee | |
| 217364 | pAcBac1-BacMam-4x-PyOtR | PyOtR | M. mazei | azidolysine (AzK) | Mammalian | TAG | Abhishek Chatterjee | |
| 218764 | pEvoltac-EcW-TGA-h14 | TrpRS-h14 (S8A, V144G, V146C) | E. coli | Bacterial | TGA | Abhishek Chatterjee | ||
| 218765 | pEvoltac EcW-TGA-h9 | TrpRS-h9 (S8A, V144S, V146A) | E. coli | Bacterial | TGA | Abhishek Chatterjee | ||
| 218766 | pEVOL-tac-EcTyrRS-VSMA*-lpp-tRNA_CUA^EcTyr | TyrRS (Y37V, D167G, D182S, F183M, L186A, D265R) | E. coli | Bacterial | TAG | Abhishek Chatterjee | ||
| 218767 | pEVOL-tac-EcTyrRS-pAAFRS-9-lpp-tRNA_CUA^EcTyr | TyrRS (Y37G, L71V, D182C, F183Y, L186C, D265R) | E. coli | Bacterial | TAG | Abhishek Chatterjee | ||
| 220800 | EF1a-L274GMmMetRS-T2A-mCherry | MetRS | M. musculus | azidonorleucine (Anl) | Mammalian | Elizabeth Kirby | ||
| 223508 | pCMV_NESPylRS(AF)_hU6tRNAPyl | PylRS (Y306A, Y384F) | M. mazei | Mammalian | TAG | Edward Lemke | ||
| 223509 | pCMV_NESPylRS(WT)_hU6tRNAPyl | PylRS | M. mazei | Mammalian | TAG | Edward Lemke | ||
| 223511 | pEVOL_PylRS(AF)-tRNA | PylRS (Y306A, Y384F) | M. mazei | Mammalian | TAG | Edward Lemke | ||
| 223512 | pEVOL_PylRS(WT)-tRNA | PylRS | M. mazei | Mammalian | TAG | Edward Lemke |
Browse Strains
The table below highlights bacteria strains that have been modified to enhance non-standard amino acid incorporation.
| ID | Strain | Description | PI | |
|---|---|---|---|---|
| 48998 | C321.ΔA | all TAG sites changes to UAG, RF1 function removed | George Church | |
| 48999 | C321 | all TAG sites changes to UAG | George Church | |
| 49018 | C321.ΔA.exp | all TAG sites changes to UAG, RF1 function removed, MutS restored so decreased mutation rate | George 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. Also see Strain 192872. | Jesse Rinehart | |
| 69493 | MCJ.559 | TAG sites, RF1 function removed, genomic deletions for improved incorporation | Michael Jewett | |
| 69495 | rEc.13.delA | 13 amber sites changed to TAA, RF1 function removed | Michael Jewett | |
| 73581 | C321.deltaA (Isaacs lab) | all TAG sites changes to UAG, RF1 function removed | Farren Isaacs | |
| 87359 | C321.∆A.opt | all TAG sites changes to UAG, RF1 function removed, improved doubling time. | George Church | |
| 98564 | C321.Ub-UAG-sfGFP | all TAG sites changes to UAG, RF1 function removed, with Ubiquitin-UAG-sfGFP reporter | George Church | |
| 98565 | C321.ΔClpS.Ub-UAG-sfGFP | all TAG sites changes to UAG, RF1 function removed, ClpS inactivated, with Ubiquitin-UAG-sfGFP reporter | George Church | |
| 174513 | Syn61 | No TCG, TCA, or TAG codons in all open reading frames | Jason W Chin | |
| 174514 | Syn61Δ3(ev5) | No TCG, TCA, or TAG codons. Deletion of serT, serU, and prfA genes | Jason W Chin | |
| 189857 | Syn61Δ3(ev5) ΔrecA (ev1) | No TCG, TCA, or TAG codons. Deletion of serT, serU, prfA, and recA genes | George Church | |
| 192872 | rEcoli XpS | All 321 UAG codons changed to UAA. mutS+, λ-, ΔybhB-bioAB, ΔprfA, ΔserB mutations for improved yield and growth | Jesse Rinehart | |
| 197655 | B95(DE3) ΔA ΔfabR ΔserB | 95 endogenous TAG codons mutated to TAA, RF1 (prfA) deleted, fabR spontaneously mutated, serB deleted for phosphoserine GCE. For expressing phosphoserine-containing proteins using genetic code expansion without buildup of prematurely truncated protein. | Ryan Mehl | |
| 197656 | BL21(DE3) ΔserC | serC gene knocked out; for expressing proteins containing site-specific non-hydrolyzable phosphoserine | Ryan Mehl | |
| 218768 | ATMW1 | Removed endogenous EcTrpRS/ttRNA pair (knocked out trpS and trpT genes). Expresses yeast TrpRS/tRNA pair instead. For directed evolution of the liberated EcTrpRS/tRNA pair to incorporate other Uaas (derived from EcNR1 strain) | Abhishek Chatterjee | |
| 218769 | ATMW-BL21 | Removed endogenous EcTrpRS/ttRNA pair (knocked out trpS and trpT genes). Expresses yeast TrpRS/tRNA pair instead. For directed evolution of the liberated EcTrpRS/tRNA pair to incorporate other Uaas (derived from BL21 strain) | Abhishek Chatterjee | |
| 218770 | ATMY1 | Removed endogenous EcTyrRS/tRNA pair (tyrS gene knocked out, tyrTV gene replaced by tolC, and tyrU gene replaced by GentR). Expresses archaeal MjTyrRS/tRNA pair instead. For directed evolution of the liberated EcTyrRS/tRNA pair to incorporate other Uaas (derived from EcNR1 strain) | Abhishek Chatterjee | |
| 218771 | ATMY3 | Removed endogenous EcTyrRS/tRNA pair (tyrS gene knocked out, tyrTV gene replaced by tRNA variant that suppress the TAG nonsense codon, and tyrU gene replaced by tRNA variant inactivated by the truncation of the anticodon loop). Expresses archaeal MjTyrRS/tRNA pair instead. For directed evolution of the liberated EcTyrRS/tRNA pair to incorporate other Uaas (derived from EcNR1 strain) | Abhishek Chatterjee | |
| 218772 | ATMY4 | Removed endogenous EcTyrRS/tRNA pair (tyrS gene knocked out, tyrTV and tyrU genes replaced by tRNA variant that suppress the TAG nonsense codon). Expresses archaeal MjTyrRS/tRNA pair instead. For directed evolution of the liberated EcTyrRS/tRNA pair to incorporate other Uaas (derived from EcNR1 strain) | Abhishek Chatterjee | |
| 218773 | ATMY5 | Removed endogenous EcTyrRS/tRNA pair (tyrS gene knocked out, tyrTV and tyrU genes replaced by tRNA variant inactivated by the truncation of the anticodon loop). Expresses archaeal MjTyrRS/tRNA pair instead. For directed evolution of the liberated EcTyrRS/tRNA pair to incorporate other Uaas (derived from EcNR1 strain) | Abhishek Chatterjee | |
| 218774 | ATMY-C321 | Removed endogenous EcTyrRS/tRNA pair (tyrS gene knocked out, tyrTV gene replaced by tolC, and tyrU gene replaced by GentR) and release factor 1. Expresses archaeal MjTyrRS/tRNA pair instead. For directed evolution of the liberated EcTyrRS/tRNA pair to incorporate other Uaas (derived from C321 strain) | Abhishek Chatterjee |
Browse Target Plasmids
The table below highlights plasmids that contain genes with modified codons for unnatural amino acid incorporation.
| ID | Plasmid | Gene/Insert | Vector Type | PI |
|---|