Part:BBa_K2916059

Toehold for Bois Noir 2.1

This toehold allows the Ribosome to bind in the RNA sequence only when there is presence of RNA related to the Bois Noir disease.

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.

Figure : Secondary structure.

Contribution: New documentation from Evry_Paris-Saclay 2020

This part is a toehold switch sensor for sequence-based detection of Bois noir. It targets a fragment of the PlsC gene of PCandidatus phytoplasma solani. 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.

Contribution: Improved part from Tec-Monterrey 2021 and prior characterization

An improved version of this part (BBa_K4092015) has been designed by Tec-Monterrey 2021 team using the Toehold Switch Creator. The improved part is designed to detect the same target sequence from Candidatus phytoplasma solani (PlsC gene) while having a more stable structure (MFE of -21.700001 Kcal/mol) potentially reducing the leakage expression.

Moreover, its structure has also been characterized and it has been predicted to be different than originally stated.

Fig. Secondary structure of original part (left) and improved one (right).

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 96
  
• 1000
  COMPATIBLE WITH RFC[1000]