RS/tRNA Foundational Publication Support
Zhang, Meng, Shixian Lin, Xinwen Song, Jun Liu, Ye Fu, Xi Ge, Xinmiao Fu, Zengyi Chang, and Peng R Chen. (2011) 2011. “A Genetically Incorporated Crosslinker Reveals Chaperone Cooperation In Acid Resistance.”. Nature Chemical Biology 7 (10): 671-7. doi:10.1038/nchembio.644.
RS/tRNA Pair Development Year
2011
ncAA(s) Incorporated
(3-(3-Methyl-3H diazirine-3-yl)-propaminocarbonyl- Nε-L-lysine ( DiZPK)
ncAA Structure (png, jpg, jpeg)
ncAA Utility
Photo-cross-linker
RS Organism of Origin
Parent RS
RS Mutations
L274A
C313S
C313S
tRNA Organism of Origin
Parent tRNA
tRNA Anticodon
CUA
Other tRNA Mutations
n/a
RS/tRNA Availability
Can be bought from AddGene Plasmid #91705 (In a pSupAR vector backbone with a Cm resistance gene)
And Plasmid #91706 (Same RS but designed to work in mammalian cells with a pCMV vectror backbone)
And Plasmid #91706 (Same RS but designed to work in mammalian cells with a pCMV vectror backbone)
Used in what cell line?
RS/tRNA Additional Notes
DiZPKRS has been evolved using Double Sieve life-death selections in DH10B cells at 1Mm DiZPK concentration. The RS was used to incorporate DiZPK in myoglobin99TAG in E.coli BL21 (DE3) with the incorporation confirmed via ESI-MS. The final yield of Myoglobin-K99DiZPK was about 9 mg/L.
In Mammalian cells, DiZPK containing full length EGFP in CHO cells with the codon for residue 37 changed to an amber codon at a DiZPK concentration of 1 Mm. The transfected cells were incubated at 37 °c with and without 1 mM DiZPK for 16 h before being visualized by a fluorescence microscope confirming incorporation.
In Mammalian cells, DiZPK containing full length EGFP in CHO cells with the codon for residue 37 changed to an amber codon at a DiZPK concentration of 1 Mm. The transfected cells were incubated at 37 °c with and without 1 mM DiZPK for 16 h before being visualized by a fluorescence microscope confirming incorporation.