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Expanded Genetic Code Icon 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:

  1. A plasmid expressing the tRNA and its cognate aminoacyl-tRNA-synthetase (aaRS) that has been evolved to incorporate non-canonical amino acids (ncAAs).

  2. A plasmid containing the gene of interest with the modified codon (typically the amber codon) that is recognized by the cognate charged tRNA.

  3. Once these plasmids have been introduced in the cells, the non-canonical amino acid can be incorporated using the existing protein translation machinery.

Expanded Genetic Code_grey.jpg

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:

  1. The non-canonical amino acid, which is generally introduced in the media.
  2. 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.
  3. A tRNA that recognizes this codon.
  4. 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 Schultz
31190 pEVOL-pBpF p-benzoylphenylalanine RS Methanocaldococcus janaschii p-benzoyl-l-phenylalanine E. coli TAG Schultz
48696 pANAP AnapRS E. coli fluorescent AA, Anap Mammalian TAG Schultz
48215 pULTRA-CNF tyrosyl synthetase Methanocaldococcus janaschii para-cyanophenylalanine (pCNPhe) E. coli TAG Schultz
49086 pDULE-ABK pyrrolysyl tRNA sythetase Methanosarcina barkeri aliphatic diazirine amino acid E. coli and Mammalian cells TAG Schultz
50831 pAcBac2.tR4-OMeYRS/GFP* tyrosyl-tRNA synthetase E. coli various unnatural amino acids Mammalian TAG Schultz
50832 pAcBac1.tR4-MbPyl pyrrolysyl-tRNA synthetase Methanosarcina barkeri variety of unnatural amino acids Mammalian TAG Schultz
68292 SepOTSλ SepRS9 E. coli Sep E. coli TAG Rinehart
51401 pAM1 NLL-MetRS E. coli azi-donorleucine (Anl) Y. enterocolitica ATG Tirrell
63177 pMarsL274G methionyl-tRNA synthetase (L274GMmMetRS) murine mutant azidonorleucine (Anl) Mammalian ATG Tirrell
64915 pMAH-POLY tyrosyl synthetase E. coli pBof Mammalian TAG Ai
73546 pEvol-pAzFRS.2.t1 pAzFRS.2.t1 E. coli p-azido-l-phenylalanine (pAzF) E. coli TAG Isaacs
73547 pEvol-pAzFRS.1.t1 pAzFRS.1.t1 E. coli p-azido-l-phenylalanine (pAzF) E. coli TAG Isaacs
62598 pKPY514 phenylalanyl-tRNA synthetase subunit E. coli p-azido-L-phenylalanine (Azf) C. elegans ATG Tirrell
62599 pKPY197 phenylalanyl-tRNA synthetase (CePheRS) C.elegans p-azido-L-phenylalanine (Azf) C. elegans ATG Tirrell
71403 pCMV-DnpK pyrrolysyl-tRNA-synthetase Methanosarcina barkeri N6‐(2‐(2,4‐dinitrophenyl)acetyl)lysine (DnpK) E. coli and Mammalian TAG Ai
71404 pMAH2-CageCys leucyl-tRNA-synthetase E. coli photocaged cysteine Mammalian TAG Ai
73544 pEvol-pAcFRS.2.t1 pAcFRS.2.t1 E. coli p-acetyl-l-phenylalanine (pAcF) E. coli TAG Isaacs
73545 pEvol-pAcFRS.1.t1 pAcFRS.1.t1 E. coli p-acetyl-l-phenylalanine (pAcF) E. coli TAG Isaacs
82417 pUltra-sY sY-specific aaRS Methanocaldococcus janaschii sulfotyrosine (sY) E. coli and rice TAG Liu
85484 pDule-tfmF A65V S158A tri-fluoromethyl-phenylalanine synthetase Methanocaldococcus janaschii family of [19]F-UAAs E. coli TAG Mehl
85494 pDule-pCNF para-cyanophenylalanine synthetase Methanocaldococcus janaschii azidoPhenylalanine E. coli TAG Mehl
85495 pDule2-pCNF para-cyanophenylalanine synthetase Methanocaldococcus janaschii azidoPhenylalanine E. coli TAG Mehl
85496 pDule-Tet2.0 Tetrazine2.0 tRNA synthetase Methanocaldococcus janaschii Tetrazine 2.0 E. coli TAG Mehl
85497 pDule2-Tet2.0 Tetrazine2.0 tRNA synthetas Methanocaldococcus janaschii Tetrazine 2.0 E. coli TAG Mehl
85498 pDule-3-nitroTyrosine (5B) 3NY (5B) synthetase Methanocaldococcus janaschii 3-nitroTyrosine E. coli TAG Mehl
85499 pDule2-3-nitroTyrosine (5B) 3NY (5B) synthetase Methanocaldococcus janaschii 3-nitroTyrosine E. coli TAG Mehl
85500 pDule-IBBN (G2) IBBN (G2) synthetase Methanocaldococcus janaschii 4-(2′-bromoisobutyramido)-phenylalanine (IBBN) and structurally analogous amino acids E. coli TAG Mehl
85501 pDule2-IBBN (G2) IBBN (G2) synthetase Methanocaldococcus janaschii 4-(2′-bromoisobutyramido)-phenylalanine (IBBN) and structurally analogous amino acids E. coli TAG Mehl
85502 pDule-para-aminoPhe pAF synthetase Methanocaldococcus janaschii para-aminoPhe E. coli TAG Mehl
85503 pDule2-para-aminoPhe pAF synthetase Methanocaldococcus janaschii para-aminoPhe E. coli TAG Mehl
89189 pMaRSC methionyl-tRNA synthetase (L274GMmMetRS) murine mutant azidonorleucine (Anl) Mammalian ATG Tirrell
91705 pSupAR-MbPylRS(DiZPK) pyrrolysyl-tRNA synthetase Methanosarcina barkeri photocrosslinkers DiZPK, DiZSeK, or DiZHSeC E. coli TAG Chen
91706 pCMV-MbPylRS(DiZPK) pyrrolysyl-tRNA synthetase Methanosarcina barkeri photocrosslinkers DiZPK, DiZSeK, or DiZHSeC Mammalian TAG Chen
92047 pCOTS-pyl-GFP(35TAG) Pyrrolysyl tRNA synthetase Methanosarcina mazei cyanobacterial TAG Alfonta
92048 gCOTS-pyl Pyrrolysyl tRNA synthetase Methanosarcina mazei cyanobacterial TAG Alfonta
99222 pTECH-chPylRS(IPYE) Pyrrolysyl tRNA synthetase Chimeric p-iodo-L-phenylalanine E. coli TAG Soll
104069 pTECH-chAcK3RS(IPYE) AcK3RS Chimeric Nε-acetyl-L-lysine E. coli TAG Liu
104070 pTECH-MbAcK3RS(IPYE) AcK3RS Methanosarcina barkeri Nε-acetyl-L-lysine E. coli TAG Liu
104071 pTECH-MmAcK3RS(IPYE) AcK3RS Methanosarcina mazei Nε-acetyl-L-lysine E. coli TAG Liu
104072 pTECH-MbPylRS(IPYE) Pyrrolysyl tRNA synthetase Methanosarcina barkeri m-iodo-L-phenylalanine E. coli TAG Liu
104073 pTECH-MmPylRS(IPYE) Pyrrolysyl tRNA synthetase Methanosarcina mazei m-iodo-L-phenylalanine E. coli TAG Liu
105829 pIRE4-Azi Azi-tRNA synthetase (EAziRS) humanized p-Azido-phenylalanine (Azi) Mammalian TAG Coin
105830 pNEU-hMbPylRS-4xU6M15 Pyrrolysyl tRNA synthetase Methanosarcina barkeri Pyl-like click amino acids, tRNA M15 Mammalian TAG Coin
113644 pRF0G-Tyr tyrosine tRNA synthetase Methanococcus jannaschii tyrosine E. coli TAG Barrick
113645 pRF0G-IodoY iodotyrosine tRNA synthetase Methanococcus jannaschii iodotyrosine E. coli TAG Barrick

Browse Strains

The table below highlights bacteria strains that have been modified to enhance non-standard amino acid incorporation.

   ID Strain Description PI
48999 C321 all TAG sites changes to TAA Church
48998 C321.ΔA all TAG sites changes to TAA, RF1 function removed Church
49018 C321.ΔA.exp all TAG sites changes to TAA, RF1 function removed, MutS restored so decreased mutation rate Church
98564 C321.Ub-UAG-sfGFP all TAG sites changes to TAA, RF1 function removed, with Ubiquitin-UAG-sfGFP reporter Church
98565 C321.ΔClpS.Ub-UAG-sfGFP all TAG sites changes to TAA, RF1 function removed, ClpS inactivated, with Ubiquitin-UAG-sfGFP reporter Church
68306 C321.ΔA all TAG sites changes to TAA, 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 TAA, RF1 function removed Isaacs
87359 C321.∆A.opt all TAG sites changes to TAA, 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