Part:BBa_K3453083
Grapevine VvTrnL-UAA Trigger 'long'
This part is a trigger for sequence-based detection of grapevine. It is a fragment of the TrnL-UAA gene of Vitis vinivera.
It was designed by the EPFL 2019 iGEM team and its sequence was deduced from “Amplification” page of the EPFL 2019 wiki where is described how the triggers were designed: the grapevine trigger was PCR amplified using as template the Vitis vinifera TrnL-UAA and the forward primers EC_fwd1 or EC_fwd1_T7 and the EC_rev1 reverse primer (it should be noted that the sequences of all reverse primers listed in the additional materials of this page are given as 3’-5’ sequences and not in the conventional 5’-3’ orientation).
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 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.
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_K3453183.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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