Part:BBa_K1586003

EsxB toehold switch J23100

Usage and Biology

A toehold switch is a type of RNA molecule known as a riboregulator/riboswitch. It is able to detect the presence of a specific ssRNA molecule (termed the 'trigger RNA') which has a sequence complementary to its switch region through base pairing. If the correct RNA molecule is detected, the protein coding region of the toehold is expressed.

A toehold switch is unique in comparison to other types of riboswitches as it is completely synthetic, and therefore easier to engineer and standardise. The fact that the toehold switch can be modified means that the switch region can be easily changed to detect any given trigger RNA molecule, and the protein coding region can be swapped for any desired reporter protein most suitable for its application.

As shown by Exeter iGEM 2015, toehold switches can be used to detect specific RNA molecules in a cell-free system. In addition to this, the plasmid DNA encoding the toehold switch can be transformed into cells in order to ascertain whether a gene is being expressed (through detection of its mRNA). The applications of this technology can range from a research tool (e.g. detection of secreted RNA in cell supernatant, detection of gene expression, etc.), through to more commercial/medical applications such as diagnostic testing.

The Part

Part K1586003 encodes for an adaptable and standardised synthetic toehold switch. This toehold switch was built to contain a standard promoter (J23100), RBS (B0032), and GFP (E0040). It was designed to be used in a diagnostic test, with an initial target of the EsxB gene mRNA. EsxB encodes for Esat6 and is involved in the pathogenicity of Mycobacterium bovis. For more on this issue and the use of toehold switches in diagnostic testing, visit Exeter iGEM 2015's wiki (http://2015.igem.org/Team:Exeter).

Characterisation

"In silico":
As well as generating experimental data in the lab, "in silico" data was also generated through the software package NUPACK (http://www.nupack.org/) using free energies. For a full explanation of how this works, please visit http://2015.igem.org/Team:Exeter/Modeling.

The equilibrium concentrations of the different components in our system were determined, along with free energy data for the different structures; the unbound trigger, the unbound toehold and the complex of the two.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 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 793