Part:BBa_K3453183

Grapevine VvTrnL-UAA Trigger 'long' transcription unit

This part is a transcription unit of the Grapevine VvTrnL-UAA Trigger 'long' (BBa_K3453083) for sequence-based detection of grapevine. The transcription is controlled by the T7 promoter (BBa_K2150031) and the strong SBa_000587 synthetic terminator (BBa_K3453000).

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.

This part is a transcription unit of BBa_K3453083 which 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 this part.

To improve the functions of these 5 grapevine toehold switches, we designed 2 new toehold switches (BBa_K3453079 and BBa_K3453080) having the trigger binding sites in the correct orientation and showed experimentally that they are able to respond to an RNA molecule transcribed from this part which has the transcription orientation of the grapevine TrnL-UAA gene.

This part proved to be functional: the transcribed grapevine RNA was able to bind to the toehold switches BBa_K3453079 and BBa_K3453080 and a readable output was generated. As expected, the release of translation inhibition was not possible with any of the toehold switches having the trigger binding sites not in a compatible orientation (BBa_K2916050, BBa_K3453073 and BBa_K3453076).

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]