This year, the focus of our project is on eradicating the outdated screening procedures now used for illnesses affecting women's health. Our approach uses multiplexed regulation, a synthetic amalgamation circuit with four toehold switches for each of the four conditions (PCOS, Endometriosis, Breast Cancer, and Ovarian Cancer), which each test for three microRNAs. The switches have genetic 'AND' gates, which provide high specificity. With the use of the collaborative database we have begun, our research is unique in that it permits the miRNA binding sites to be switched around.
One blood sample can be used to test for many miRNAs simultaneously, and the findings are available much faster than with earlier techniques. Given the presence of the correct miRNA, the toehold switches will produce various fluorescent proteins once they have unfolded. The chosen fluorescent proteins can be evaluated independently because they have different wavelengths.
We have decided to use miRPA to amplify the miRNA extracted from our sample in order to improve the accessibility of our test. This eliminates the need for an expensive thermocycler by enabling the miRNA to be isothermally amplified.
Project GENOSWITCH’s primary aim is to develop a genetic engineering-based cell-free modular test for microRNA (miRNA) biomarkers that can simultaneously detect cases considerably earlier than current diagnostic methods, allowing patients' symptoms to be monitored by their doctor and medicine to be administered.
Many teams in the past have submitted toehold switches with only one input such as the 2017 BATMAN project (Part:BBa_K2206000). The 2021 RIBOTOX project developed an AND-gate switch which can detect two RNAs simultaneously. The ability to detect two RNAs for one condition meant that luciferase was only produced when two trigger RNAs were present at sufficient concentrations which helped to increase the specificity. For other prevalent conditions however, this could prove problematic if multiple strands need to be detected as it is difficult to monitor and create the hardware to help detect a conglomerate of RNA. Furthermore, switches which induce a reporter protein in a CDS are unspecific. This is because the same miRNAs are upregulated in multiple conditions as extra-cellular microRNA networks are complex. We were able to scale up the design of past iGEM teams to produce systems that are able to detect several microRNAs. This exponentially increased specificity to the conditions we were testing for.
The secondary goal involved developing a framework to help researchers and iGEM teams develop tests for various conditions at low miRNA concentrations in the future and a software tool that is easy to understand to increase the use of in-silico experimentation and reduce lab costs. Our software is designed to make the lives of other teams’ members easier by giving them a quick and easy way of designing our high-specificity, Gen4 toehold switches for any given miRNA strands without the need for complex thermodynamic calculations. The software utilises the NUPACK python library for its calculations.
It is being shipped via appimages and ships with a beta version of NUPACK that we are allowed to distribute. It also has a front-end UI that is easy to read and navigate alongside offering many quality of life features such as full-integration with the latest version of miRBase (so long as the user is connected to the internet), support for importing .fasta files and many ways to export the results: This includes visually seeing the results in the app as either a polymer graph or equilibrium base pair probability matrix. Results can also be exported to a text file for a detailed analysis of all structures and their statistics or to a .fasta file containing only the strands generated by the software. All of these features offer a convenient way for future iGEM teams to use our toehold switch designs in any desired context.
The final objective was to raise awareness about women's health and the specific illnesses that are related to it. These conditions are largely unknown while being common and frequently have a negative impact on lives in both developed and developing countries. We have carefully considered how our sensor might be applied in a clinical environment as a component of our work in human practises. As part of our integrated human practises, we spoke with numerous clinicians and established a plan for how our test might be utilised in screening programmes. On their advice, we have also increased our sensor's sensitivity and specificity. We have also looked into the potential detection of other diseases by our sensor and the advantages of our approach.
In a clinical implementation, miRNAs extracted from blood serum would be added to a cell free system containing an excess of our amalgamation. The unique toehold switches would detect specific miRNAs and regulate translation of the corresponding fluorescent protein in response. The intensity of fluorescence produced by our circuits is therefore indicative of the miRNA levels in the body fluid. To help reduce costs, we designed and built a £4 combined fluorometer and densitometer, coined a luminometer, to cheaply quantify the fluorescence from our circuit without a plate reader. This allows our test to be used in the field and in less developed countries.