RS/tRNA Pair Development Year
2023
RS Organism of Origin
Parent RS
RS Mutations
E412P
E414F
P495M
I496W
F529S
E414F
P495M
I496W
F529S
tRNA Organism of Origin
Parent tRNA
tRNA Anticodon
CUA
Other tRNA Mutations
C2U
G4C
G6C
G29A
A31U
G34C
C35U
G37A
U39A
C40U
C41U
C67G
C69G
G71A
G4C
G6C
G29A
A31U
G34C
C35U
G37A
U39A
C40U
C41U
C67G
C69G
G71A
RS/tRNA Availability
Entire kit for the "PermaPhos" system (nhpSer incorporation using GCE) available as Addgene kit #1000000226.
Component Addgene IDs are:
201922 - machinery plasmid, including RS/tRNA pair
174075 and 174076 - plasmids for control sfGFP forms which can also be used for other POI
201923 - plasmid for Frb pathway
197656 - E. coli strain BL21(DE3) ΔserC
Component Addgene IDs are:
201922 - machinery plasmid, including RS/tRNA pair
174075 and 174076 - plasmids for control sfGFP forms which can also be used for other POI
201923 - plasmid for Frb pathway
197656 - E. coli strain BL21(DE3) ΔserC
Used in what cell line?
RS/tRNA Additional Notes
Using this RS/tRNA pair for nhpSer incorporation along with a biosynthetic path nhpSer was reported by Zhu et al. (2023). However the GCE tools SepRS2, Sep-tRNA-v2, and EF-Sep (a variant of EF-Tu) were developed in foundational 2015 and 2017 and 2011 papers, respectively. To work well with nhpSer it is essential to include a plasmid that encodes enzymes from the Frb pathway that lead to the endogenous biosynthesis of nhpSer.
Single and double high-fidelity nhpSer incorporation into sfGFP (site 150) yielded ∼120 and 30 mg/L culture, respectively, corresponding to efficiencies of ~40% and 13%. This is ~40-fold higher than nhpSer incorporation using exogenous 2 mM nhpSer as opposed to the endogenous biosynthesis.
Single and double high-fidelity nhpSer incorporation into sfGFP (site 150) yielded ∼120 and 30 mg/L culture, respectively, corresponding to efficiencies of ~40% and 13%. This is ~40-fold higher than nhpSer incorporation using exogenous 2 mM nhpSer as opposed to the endogenous biosynthesis.