RNA

Part:BBa_K1499251

Designed by: Raman Nelakanti   Group: iGEM14_StanfordBrownSpelman   (2014-08-27)

supP tRNA

This part encodes the amber suppressing tRNA supP, a mutated form of the leucine tRNA LeuX.

Usage and Biology

There have been a variety of tRNA mutations found in nature that allow translation of stop codons. These are known collectively as "nonsense suppressors" because the bacteria they are found in are susceptible to infection by viruses with nonsense mutations. This leucine tRNA supP differs from the endogenous leucine tRNA LeuX only in the anticodon and is able to recognize the amber stop codon (UAG) and insert a leucine at that position.[1] This part contains the supP tRNA along with 100 bases upstream and 50 bases downstream to allow for proper tRNA expression and processing.

This part is used in Stanford-Brown-Spelman's 2014 iGEM project to create the Amberless Codon Security system (Figure 1).

Figure 1. Our approach to the Amberless Codon Security. By recoding the UAG stop codon to translate into an amino acid, only cells that have a tRNA with the anticodon AUC will produce the complete protein. In our experiment, we used a tRNA that charges with leucine to translate the UAG codon.


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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

Verification

The part was sequence verified in the pSB1C3 backbone before submission to the registry. One forward read was obtained using VF2.

[[Image:|700px|thumb|center|Figure 2. GFP with two amber stop codons matches the expected sequence.]]

Results

This part was verified to work by the GFP and aeBlue generator constructs. See this part's experience page for these results.

References

1. Thorbjarnardóttir, S et al. (1985) Leucine tRNA family of Escherichia coli: nucleotide sequence of the supP(Am) suppressor gene. J. Bacteriol. 161: 219–22. PMID: 2981802.

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Categories
Parameters
chassisAmberless E. coli (C321.ΔA)