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.

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
	48215	pULTRA-CNF	tyrosyl synthetase	Methanocaldococcus janaschii	para-cyanophenylalanine (pCNPhe)	E. coli	TAG	Schultz
	48696	pANAP	AnapRS	E. coli	fluorescent AA, Anap	Mammalian	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
	73546	pEvol-pAzFRS.2.t1	pAzFRS.2.t1	E. coli	p-azido-l-phenylalanine (pAzF)	E. coli	TAG	Isaacs
	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
	73544	pEvol-pAcFRS.2.t1	pAcFRS.2.t1	E. coli	p-acetyl-l-phenylalanine (pAcF)	E. coli	TAG	Isaacs
	73547	pEvol-pAzFRS.1.t1	pAzFRS.1.t1	E. coli	p-azido-l-phenylalanine (pAzF)	E. coli	TAG	Isaacs
	89189	pMaRSC	methionyl-tRNA synthetase (L274GMmMetRS)	murine mutant	azidonorleucine (Anl)	Mammalian	ATG	Tirrell
	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
	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
	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
	122650	Mm-PylRS-AF/Pyl-tRNACUA	Pyrrolysyl tRNA synthetase	Methanosarcina mazei	trans-cyclooct- 2-ene-lysine (TCOK)	Mammalian	TAG	Hang
	126035	pEVOL-ABK	pyrrolysyl tRNA sythetase	Methanosarcina barkeri	3’-azibutyl-N-carbamoyl-lysine (AbK)	E. coli	TAG	Musacchio
	127411	pEVOL-pylT-N346A/C348A	pyrrolysyl tRNA sythetase	Methanosarcina mazei	mono-substituted phenylalanine derivatives and tyrosinyl ethers	E. coli	TAG	Liu
	137908	PylRS-AS	pyrrolysyl tRNA sythetase	Methanosarcina mazei	fluorophenylalanine derivatives	E. coli	TAG	Liu
	137976	pEVOL-AckRS	pyrrolysyl tRNA sythetase	Methanosarcina mazei	AzHeK	E. coli	TAG	Liu
	140009	pAS_4xMma PylT_FLAG-Mma PylRS	pyrrolysyl tRNA sythetase	Methanosarcina mazei	CpK, AbK	Mammalian	TAG	Elsaesser
	140011	pAS_4xMx PylT_FLAG-Mx PylRS	pyrrolysyl tRNA sythetase	Methanomethylophilus alvus Mx1201	CpK	Mammalian	TAG	Elsaesser
	140013	pAS_4xMx PylT C41CA_FLAG_Mx PylRS	pyrrolysyl tRNA sythetase	Methanomethylophilus alvus Mx1201		Mammalian	TAG	Elsaesser
	140014	pAS_4xMx PylT C41CA_FLAG_Mx PylRS YA	pyrrolysyl tRNA sythetase	Methanomethylophilus alvus Mx1201		Mammalian	TAG	Elsaesser
	140018	pAS_4xBstTyrT(CUA)_EcoTyrRS-FLAG	tyrosine tRNA synthetase	E. coli		Mammalian	TAG	Elsaesser
	140019	pAS_4xEcoLeuT(CUA)_AnapRS	AnapRS	E. coli	azido-phenylalanine	Mammalian	TAG	Elsaesser
	140021	pAS_4xMma PylT_FLAG-AcKRS	AcKRS	Methanosarcina mazei	acetyl-lysine	Mammalian	TAG	Elsaesser
	140022	pAS_4xMma PylT_FLAG-PcKRS	PcKRS	Methanosarcina barkeri	photocaged-lysine	Mammalian	TAG	Elsaesser

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 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