Part:BBa_K2916051

Toehold for Grapevine Endogenous Control 2.1

Usage and Biology

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.

Improvement by Evry_Paris-Saclay 2020

This part is a toehold switch sensor for sequence-based detection of grapevine. It targets a fragment of the TrnL-UAA gene of Vitis vinifera. 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.

This part has been improved to be functional. We have successfully built two parts, the Grapevine Switch 3.1 (BBa_K3453079) and the Grapevine Switch 3.2 (BBa_K3453080), which are able to act as toehold switches and (i) efficiently repress the downstream reporter gene expression in the absence of the cognate trigger and (ii) release the translation inhibition in the presence of the cognate trigger, an RNA molecule having the transcription orientation of the TrnL-UAA gene of grapevine.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 53