Part:BBa_K2916065
Toehold for Flavescence Doree 2.2
Toehold switches are synthetic riboregulators that regulate the expression of a downstream gene. They sequester the Ribosome Binding Site (RBS) and Start Codon by forming a secondary structure called a hairpin structure in its OFF state (acting as a “lock” for protein expression). A second strand of mRNA, complementary to the trigger binding region, needs to be present to activate the toehold. It will bind to that region and linearize the hairpin structure, freeing the RBS for the ribosomes to bind and allowing for translation of the downstream gene. Our toehold switches function at 37°C, and protein production is carried out in a cell-free PURE system. Reference Toehold switches: de-novo-designed regulators of gene expression, Green et al., 2014 In the encoding part, we have a toehold region, a hairpin region which contains a stem and a loop, a refolding region and a spacer. In design, it is crucial to exclude any ending codon in the refolding and spacer region. The refolding region allows ribosomes to access the protein coding region with less energy, thus increasing the kinetics of the reaction.
Contribution: New documentation from Evry_Paris-Saclay 2020
This part is a toehold switch sensor for sequence-based detection of Flavescence Doree. It targets a fragment of the 16S ribosomal RNA gene of Candidatus phytoplasma vitis. However, the trigger binding site is in the same orientation as in the transcriptional orientation of its target gene. Thus it is unlikely that this toehold switch will be able to detect the presence of an RNA naturally expressed in a cell.
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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