Part:BBa_K3453082

Grapevine VvTrnL-UAA Trigger 'short reverse'

This part is a trigger for sequence-based detection of grapevine. It is a fragment of the TrnL-UAA gene of Vitis vinivera in the reverse orientation compared to BBa_K3453081.

Usage and Biology

A toehold switch is an RNA–based device containing a ribosome binding site (RBS) and an ATG start codon embedded in a hairpin structure that blocks translation initiation [1]. The hairpin can be unfolded upon binding of a trigger RNA thereby exposing the RBS and the ATG start codon permitting translation of the reporter protein (Figure 1).

Figure 1. Toehold switches principle.

BBa_K3453081 was designed by the EPFL 2019 team as a trigger for 5 of their toehold switches: BBa_K2916050, BBa_K2916051, BBa_K2916052, BBa_K2916053 and BBa_K2916054. However, these 5 toehold switches do not have the trigger binding sites in the correct orientation and thus are not able to be activated by the RNA transcribed from BBa_K3453081. However, the hairpin structures of these 5 grapevine toehold switches are predicted to be unfolded by this part which is the reverse complement of BBa_K3453081.

We tested the functionality of this part under controlled of the T7 promoter (BBa_K2150031) and the strong SBa_000587 synthetic terminator (BBa_K3453000) in the composite part BBa_K3453182.

This part proved to be functional: the transcribed grapevine RNA was able to bind to the toehold switches BBa_K2916050, BBa_K3453073 and BBa_K3453076 and a readable output was generated. As expected, the release of translation inhibition was not possible with the toehold switch having the trigger binding sites not in a compatible orientation (BBa_K3453079).

Full results are available on the BBa_K2916050, BBa_K2916068, BBa_K3453170, BBa_K3453171, BBa_K3453172, BBa_K3453173, BBa_K3453174, BBa_K3453175, BBa_K3453176, BBa_K3453177, BBa_K3453178, BBa_K3453179 and BBa_K3453180 pages in the registry.

References

[1] Green AA, Silver PA, Collins JJ, Yin P. Toehold switches: de-novo-designed regulators of gene expression. Cell (2014) 159, 925-939.

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]