Nonstandard Amino Acids

The Genomically Recoded Organism and Its Genotype

E. coli C321.∆A.exp (Lajoie, et al. 2013)
Genotype: MG1655 ∆(ybhB-bioAB)::zeoR ∆prfA
  • all 321 UAG codons changed to UAA
  • mutS+ (wild-type mutation rate)
  • λ- (growth at 37慢 not deleterious)
  • ZeoR (genome resistance to zeocin)
  • release factor RF1 deleted (amber codon termination abolished)
  • biotin auxotroph in defined media
Obtain C321.∆A.exp from Addgene

Canonical Amino Acids


Name Abbreviation Structure
L-Tyrosine
Y


Nonstandard Amino Acids and their Orthogonal Aminoacyl tRNA Synthetase/tRNA Pairs


A great majority of the non-canonical amino acids we currently use are incorporated in E. coli C321.∆A.exp using the Methanococcus jannaschii tyrosyl-tRNA synthetase/tRNA pair (MjYRS and MjYCUA) with the corresponding indicated mutations to the M. jannaschii tyrosyl tRNA synthetase gene. (Wang, et al. 2001)
Obtain pEVOL-pAzF from Addgene

Name Abbreviation Structure Canonical Analog Parent aaRS aaRS Mutations
(Rel. to parent)
tRNA Features Ref.
3-Iodo-L-tyrosine IodoY Tyrosine MjYRS H70A
D158T
I159S
D286R
MjYCUA 3
3-Amino-L-tyrosine AminoY Tyrosine MjYRS Y32Q
L65E
F108G
Q109L
D158S
L162Y
D286R
MjYCUA 4
3-Nitro-L-tyrosine NitroY Tyrosine MjYRS Y32R
H70L
Q155M
D158G
I159L
L162H
D286R
MjYCUA 5
o-Nitrobenzyl-L-tyrosine ONBY Tyrosine MjYRS Y32G
L65G
F108E
D158S
L162E
D286R
MjYCUA Photocaging ability:
nitrobenzyl caging group
cleaves with 365nm light
6
4-Azido-L-phenylalanine AzF Phenylalanine MjYRS Y32T
E107N
D158P
I159L
L162Q
D286R
MjYCUA Azide (N3) moiety
useful for click chemistry
7
4-Cyano-L-phenylalanine CNF Phenylalanine MjYRS Y32L
L65V
F108W
Q109M
D158G
I159A
D286R
MjYCUA 8
3,4-Dihydroxy-L-phenylalanine L-DOPA Phenylalanine MjYRS Y32L
A67S
H70N
A167Q
D286R
MjYCUA 9
3-Bromo-L-tyrosine BromoY Tyrosine        
3-Chloro-L-tyrosine ChloroY Tyrosine        


An additional non-canonical amino acid that we currently use with E. coli C321.∆A.exp was evolved by compartmentalized partnered replication from the Saccharomyces cerevisiae tryptophanyl-tRNA synthetase paired with a rationally designed tRNA, both from the Ellington Lab at UT Austin. (Ellefson, et al. 2013)
Name Abbreviation Structure Canonical Analog Parent aaRS aaRS Mutations
(Rel. to parent)
tRNA
5-Hydroxy-L-tryptophan 5HTP Tryptophan ScWRS T107C
P254T
C255A
(R3-13)
ScWCUA
(AS3.4)

Amino Acid Usage Notes



Amino Acid
(Abbreviation)
CAS Number Solvent MW
(g/mol)
[Stock] [Stock]
(mg/mL)
[Culture] Notes
Y 60-18-4 Water 181.19 10mM 1.812 250然 Requires heating to 70慢 to dissolve
Stock precipitates out of solution at or below room temperature
IodoY 70-78-0 Water 307.09 10mM 3.071 250然 Dissolves at room temperature with ~1 hour of shaking
AminoY (FAA) 300-34-5 196.203
AminoY (HS) 23279-22-3 Water 287.14 10mM 250然 Dissolves readily without shaking or heat
BromoY 38739-13-8 Water 260.0846 10mM 250然
ChloroY 7423-93-0 Water 215.63 10mM 250然
NitroY 621-44-3 Water 226.2 10mM 2.262 250然 Will dissolve with heating to 50慢, no shaking
ONBY (FAA) 184591-51-3 316.31 Light sensitive
ONBY (S) 207727-86-4 50% DMSO
50% Water
(exothermic)
352.77 50 mM 250然 Light sensitive
Requires adding OH- to dissolve
AzF 33173-53-4 10% DMSO
90% Water
206.204 10mM 2.062 250然 Light sensitive
Requires heating and shaking to dissolve
CNF 167479-78-9 Water 190.2 10mM 1.902 250然 Dissolves at room temperature with ~15 minutes of shaking
L-DOPA 59-92-7 Water 197.1879 10mM 1.972 250然 Oxidizes quickly and turns black in solution
5HTP 4350-09-8 Water 220.2246 10mM 2.202 250然

FAA: free amino acid
S: salt
HS: hydrated salt

References

1. Lajoie, M. J., Rovner, A. J., Goodman, D. B., Aerni, H.-R., Haimovich, A. D., Kuznetsov, G., Mercer, J. A., Wang, H. H., Carr, P.A., Mosberg, J.A, Rohland, N., Schultz, P.G., Jacobson, J.M., Rinehart, J., Church, G.M., Isaacs, F. J. (2013). Genomically recoded organisms expand biological functions. Science, 342(6156), 357360. doi:10.1126/science.1241459
2. Wang, L., Brock, A., Herberich, B., & Schultz, P. G. (2001). Expanding the genetic code of Escherichia coli. Science, 292(5516), 498500. doi:10.1126/science.1060077
3. Sakamoto, K., Murayama, K., Oki, K., Iraha, F., Kato-Murayama, M., Takahashi, M., Ohtake, K., Kobayashi, T., Kuramitsu, S., Shirouzu, M., Yokoyama, S. (2009). Genetic encoding of 3-iodo-L-tyrosine in Escherichia coli for single-wavelength anomalous dispersion phasing in protein crystallography. Structure, 17(3), 335344. doi:10.1016/j.str.2009.01.008
4. Seyedsayamdost, M.R., Xie, J., Chan, C.T., Schultz, P.G., Stubbe, J. (2007). Site-specific insertion of 3-aminotyrosine into subunit α2 of E. coli ribonucleotide reductase: direct evidence for involvement of Y730 and Y731 in radical propagation. Journal of the American Chemical Society, 129(48), 15060-15071.
5. Neumann, H., Hazen, J. L., Weinstein, J., Mehl, R. A., & Chin, J. W. (2008). Genetically encoding protein oxidative damage. Journal of the American Chemical Society, 130(12), 40284033. doi:10.1021/ja710100d
6. Deiters, A., Groff, D., Ryu, Y., Xie, J., & Schultz, P. G. (2006). A genetically encoded photocaged tyrosine. Angewandte Chemie, 45(17), 27282731. doi:10.1002/anie.200600264
7. Chin, J.W., Santoro, S.W., Martin, A.B., King, D.S., Wang, L., Schultz, P.G. (2002). Addition of p_-Azido-L-phenylalanine to the Genetic Code of _Escherichia coli. Journal of the American Chemical Society, 124 (31), 9026-9027.
8. Schultz, K. C., Supekova, L., Ryu, Y., Xie, J., Perera, R., & Schultz, P. G. (2006). A genetically encoded infrared probe. Journal of the American Chemical Society, 128(43), 1398413985. doi:10.1021/ja0636690
9. Alfonta, L., Zhang, Z., Uryu, S., Loo, J. A., & Schultz, P. G. (2003). Site-specific incorporation of a redox-active amino acid into proteins. Journal of the American Chemical Society, 125(48), 1466214663. doi:10.1021/ja038242x
10. Ellefson, J. W., Meyer, A. J., Hughes, R. A., Cannon, J. R., Brodbelt, J. S., & Ellington, A. D. (2013). Directed evolution of genetic parts and circuits by Compartmentalized Partnered Replication. Nature Biotechnology, 32(1), 97101. doi:10.1038/nbt.2714
Topic attachments
I Attachment Action Size Date Who Comment
pngpng 5htp.png manage 12.1 K 26 Jun 2015 - 23:34 Main.JordanMonk  
pngpng CNF.png manage 38.5 K 27 Jun 2015 - 00:02 Main.JordanMonk  
pngpng DOPA.png manage 5.4 K 27 Jun 2015 - 00:02 Main.JordanMonk  
pngpng Y.png manage 38.5 K 27 Jun 2015 - 00:12 Main.JordanMonk  
pngpng bromoy.png manage 63.7 K 19 Nov 2015 - 23:43 Main.JordanMonk  
pngpng chloroy.png manage 64.8 K 19 Nov 2015 - 23:43 Main.JordanMonk  
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Topic revision: r19 - 20 May 2016 - 23:43:52 - Main.JordanMonk
 
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