RS/tRNA Pair Development Year
2015
RS Organism of Origin
Parent RS
RS Mutations
E412S
E414I
P495R
I496R
K356E
N361D
L590I
E414I
P495R
I496R
K356E
N361D
L590I
tRNA Organism of Origin
Parent tRNA
tRNA Anticodon
CUA
Other tRNA Mutations
C20U
Multiple tRNAs?
In 2015, the G37A mutation was added to tRNA-Sep to make tRNA-SepA37 with improved UAG read-through. It more than doubled protein production.
In 2023, In addition to having A37, the C2:G71 base pair was changed to G2:C71 to decrease mischarging by GlyRS and ThrRS. This was called "tRNA-pSer-Opt"
In 2023, In addition to having A37, the C2:G71 base pair was changed to G2:C71 to decrease mischarging by GlyRS and ThrRS. This was called "tRNA-pSer-Opt"
RS/tRNA Availability
SepRS9, EF-Sep21 and tRNA-SepA37 are all on AddGene #68292. There are 4 copies of the tRNA on this plasmid, which may cause instability when propagating the plasmid.
The 2023 updated system used for studying peptides of the human pSer proteome expressed in E coli is Addgene #188537, and the rEcoli.XpS strain is Addgene #192872. This system expresses only 1 copy of the tRNA.
The 2023 updated system used for studying peptides of the human pSer proteome expressed in E coli is Addgene #188537, and the rEcoli.XpS strain is Addgene #192872. This system expresses only 1 copy of the tRNA.
Used in what cell line?
RS/tRNA Additional Notes
The EF-TU (EF-Sep21) has mutations H67R, E216V, D217G, F219Y, T229S, N274W to work with pSer. This lamba system yield 9-fold more GFP(site17) than the previous SepOTSα (SepRS, tRNA-Sep, EF-Sep) using C321.ΔA. Furthermore, the fidelity Sep was ~85% compared with ~50%. Also made MEK1 with pSer at sites 218 and 222 at 2 mg/L, but due to near-cognate suppression, the fidelity was only ~30% based on phostag gel analysis.