Project: MERS-CoV molecular diagnosis via the toehold switch mechanism and glucose monitoring


Detection Mechanism: Toehold Switch

  1. Developed a computational tool for predicting mRNA sequences with optimal toehold switch functionality.
  2. Successfully constructed a SynBio part in a pGEM vector, containing a toehold switch regulated by a specific and conserved MERS-CoV genomic region and encoding split trehalase - part A.

Reporter System: Trehalase & Split Trehalase

  1. Proposed an innovative reporter system for semi-quantitative diagnosis, incorporating the enzyme trehalase as well as a split trehalase system expected to interact via coiled coil interactions.
  2. Successfully predicted the chimeric protein structures of our split trehalase system (part A and part B), via Homology Modeling.
  3. In order to gain insight on the split trehalase system behaviour, we performed Molecular Dynamics Simulations, with promising results.
  4. Studied the kinetics of the cell-free system and developed a consistent kinetic model for the trehalase coupled transcription/translation and the enzymatic reaction.
  5. Tested the TreA enzymatic assay with the DNS method and with a common glucometer, both showing promising results for rapid POC diagnosis.

Applied Design: Diagnostic Kit

  1. Proposed the detailed design of a diagnostic kit for POC diagnosis, with significant competitive advantages to existing diagnostic methods (qRT-PCR).
  2. Developed a 3D model of the diagnostic kit for the study of the temperature profile, calculating proper times and dimensions for optimum performance.

Overview: Our overall achievements

  1. Monitored the cell-free trehalase-produced glucose with a commercial glucometer.
  2. Concluded that detectable trehalase amounts are produced in the time frame indicated by the kinetic model, indicating that trehalase could be a suitable reporter protein for molecular diagnostics.
  3. Demonstrated that the proposed diagnostic method can be performed in under one hour, therefore being suitable for Point-of-Care diagnosis.
  4. Successfully completed the InterLab Study

Human Practices: Engaging with the world

  1. Performed a series of educational and public-engagement-oriented activities, entitled “Synthetic Biology: The Principles of Engineering Life.”
  2. Gathered our observations on the presence of Synthetic Biology in the Greek reality in an elaborate report. Further discussed our findings with specialists and academics, and documented our suggestions.
  3. Created a video addressing high school students that took a linguistic approach on explaining Synthetic Biology.
  4. Documented the procedure of organizing a SynBio Workshop on a guide for future iGEM reference.
  5. Got in contact with specialists from all over the world and gained valuable feedback that we incorporated in our project, both in wet lab and dry lab experiments, as well as in our suggested diagnostic method and final product design.
  6. Collaborated with various iGEM-ers from around the world and engaged in meaningful discussions with the scientific community of Synthetic and Systems Biology.


  1. We encountered difficulties in transforming the trigger parts, therefore we could not assess the functionality of the constructed toehold.
  2. We only produced a toehold switch with the split trehalase as a reporter protein.
  3. The constructed toehold switch could not be inserted in the pSB1C3 vector. The ligation product was finally transformed and minipreped, however, after digestion, no bands were detectable on the gel.
  4. The Toehold Switch Molecular Dynamics simulation was an aspiration that remained fruitless, as RNA modelling is a demanding field where limited softwares are available for producing promising results.

Future Goals

  1. Design and perform a trehalase quantification assay and establish a viral load- protein production correlation.
  2. Create and test toehold switches containing the whole trehalase and evaluate this reporter protein relatively to beta-galactosidase.
  3. Apply the toehold switch detection mechanism coupled with the glucose monitoring test for a series of other viruses such as West Nile virus and coinfection cases such as Hepatitis C and B coinfection.
  4. Evaluate the potential parallel use of different toehold-switches targeting different viral genomic regions for more reliable diagnosis.
  5. Create a 3D model of the 2D studied toehold switches.
  6. Investigate other methods for the leucine zippers in silico study and the assembled trehalase molecule modeling.
  7. Validate the current model with experimental data and develop a new kinetic model for the split trehalase reporter.
  8. Build and assess a prototype of the proposed diagnostic kit.