MERS-CoV (Middle East Respiratory Syndrome Coronavirus) is a Coronavirus, endemic to the Middle East. The virus attacks the human respiratory system and is highly pathogenic, causing a series of non-specific symptoms which complicate the diagnosis. The World Health Organization has declared MERS-CoV as one of the most likely to cause a future epidemic and urges for further research. The mortality rate of humans infected by MERS-CoV is approximately 35%, making on-time diagnosis critical for treatment and epidemic prevention.
iGEM Athens 2018 team aims to develop a molecular diagnostics kit for the detection of MERS-CoV. To meet the existing societal needs, we aim for an easy-to-use, rapid test that is reliable, safe, and usable on the field – even by the untrained.
Our detection mechanism is based on the Toehold-Switch technology. Toehold switches are hairpin-shaped riboregulators that precede a protein coding sequence in a synthetic mRNA molecule. The conformation of the switch regulates the expression of the protein; in the absence of a trigger complementary RNA sequence, the switch region folds, inhibiting the binding of the ribosome to the RNA and subsequently the expression of the coding sequence. If the target sequence is present, it binds to the switch region causing its unfolding, allowing the protein production.
Incorporating this mechanism as a DNA construct in a cell-free transcription and translation system creates a robust, genetically engineered circuit that can be used as a biosensor. DNA or RNA segments of viral or pathogenic origin can provoke the unfolding of a specific toehold switch-gene complex and lead to the expression of a reporter protein, indicating the existence of the target segments. It is preferable that the reporter protein produce an easy-to-read signal, such as a change in colour.
We attempt to refine the existing technology further by testing alternative peptides as strong reporters, improving the rapidness and the sensitivity of our diagnostic test. The reporter protein has to be of a relatively small size, alleviating the transcriptional and translational load as much as possible. We will design toeholds activated by MERS-CoV sequences and regulate the expression of an engineered enzyme
The toehold switch design is assisted by bioinformatics tools suggesting the best candidate target sequences, taking into consideration the viral genome, the sample origin and the natural microbiota of the sample. On this scope, we are designing Pre.Di.C.T. (Predictive Diagnostic Custom Toehold-Switch), a user-friendly generalized workflow that will facilitate the design of molecular-diagnostics systems for future research on other viruses.
You can read the original post on the PLOS Synbio Blog: http://blogs.plos.org/synbio/2018/07/16/igem-2018-two-synbio-teams-from-greece-are-here-to-leave-their-mark/