RNA

Part:BBa_K4171009

Designed by: HUANG, YEN-JUNG   Group: iGEM22_NCKU_Tainan   (2022-08-11)


tRNA(UTuX)

Background

tRNAUTuX (BBa_K4171009) is a tRNA species with some codons of tRNASec (BBa_K4171008) mutated for higher selenocysteine (Sec) peptide insertion frequency and also better binding efficiency with SelA (BBa_K4171002)[1].

Usage

tRNAUTuX (BBa_K4171009) is used for aminoacylation with serine (Ser), which becomes seryl-tRNAUTuX, the primary substrate of SelA. Although it has the best performance in binding with SelA among the three tRNA species (BBa_K4171007, BBa_K4171008 and BBa_K4171009) we used, its reaction with wild-type SerRS (BBa_K4171001) is not that satisfying, according to a research[2].



Fig. 1. In vitro Sec synthesis pathway

Characterization



Fig. 2. Confirmation of in vitro tRNAUTuX (BBa_K4171009) synthesis by agarose gel electrophoresis. M: Marker; Lane1: tRNAUTuX (91bp)


The bands of seryl-tRNAUTuX have upshifted compared to the band of the tRNAUTuX (Fig. 3). The result shows that the amino acid was successfully charged to the tRNA.


Fig. 3. Confirmation of the result after aminoacylation by Urea PAGE. Lane1: tRNAUTuX (BBa_K4171009); Lane2: seryl-tRNAUTuX


After successful aminoacylation, serine on the tRNA was transformed into Sec. In the HPLC results, the retention time of the Sec standard is 10.5 minutes (the peaks at 3.5 minutes and 4.5 minutes are solvent peaks), which is the same as that of Sec synthesized via the in vitro pathway, indicating that both tRNA and enzymes (BBa_K4171002 and BBa_K4171003) function as expected.


Fig. 4. Confirmation of Sec by HPLC. This is the result of Sec synthesized by tRNAUTuX (BBa_K4171009).

Reference

[1] Miller C, Bröcker MJ, Prat L, et al. A synthetic tRNA for EF-Tu mediated selenocysteine incorporationin vivoandin vitro. FEBS Letters. 2015;589(17):2194-2199. doi:10.1016/j.febslet.2015.06.039

[2] Fu X, Crnković A, Sevostyanova A, Söll D. Designing seryl‐ tRNA synthetase for improved serylation of selenocysteine tRNA s. FEBS Letters. 2018;592(22):3759-3768. doi:10.1002/1873-3468.13271

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 40
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 30


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