Team:CLSB-UK/Demonstrate
Demonstrate
We set out to develop a cheap and effective test to diagnose NSCLC by measuring miRNA levels using toehold switches.
Toehold Switches
We have shown that toehold switches are capable of being used as a tool to quantify miRNA levels in a cell free system. Our sensor outputs different levels of fluorescence for varying miRNA concentrations. Additionally, our sensor was not activated when tested with non-target miRNAs.
Our modeling has shown that our second series of switches exhibit very low levels of cross-hybridisation. As such they can achieve single base mismatch specificity.
Our cost analysis shows that our sensor would provide an extremely cheap way to screen for and detect NSCLC. Combined with our cheap and effective fluorometer, and use of a sterile and abiotic cell free system, our test would allow for screening programmes in developing countries.
Take a look at our results page and cost model for more details.
Clinical implementation strategy
We developed a clinically viable implementation strategy for our sensor. Our cost model proved our strategy is cost-effective and discussions with doctors confirmed that the early detection our sensor provides will reduce NSCLC mortality rates. We have explained how our sensor meets NICE requirements for a screening tool, based on feedback from doctors. We have also selected a few other diseases, and explained how our tool could be successfully applied to them. In addition, we have explained how our sensor would be viable and highly beneficial in developing countries.
View our cost model and silver and gold integrated human practices pages for more details.
Fluorescence quantification
To prove the economic viability of a screening programme with our toehold switches, we needed to create a cheap, effective and portable fluorometer.
Our combined fluorometer and densitometer costs just $6 in parts, using low-cost, readily-available components and a small 3D-printed cuvette holder. This allows us to quantify the reporter protein regulated by our toehold switch in less developed countries and in the field.
While crucial to our proof of concept, a low cost, easy to build and powerful combined fluorometer and densitometer offers massive advantages to synthetic biology as a whole, especially for high schools or community labs who may not be able to afford a plate reader.
See our hardware and measurement pages for more details.
Model validation
We modeled our system to improve the design of our toehold switches and to analyse our results.
NUPACK allowed us to design highly specific and stable toehold switches, which were able to quantify different miRNA levels.
Our mass action kinetics model fit the experimental data extremely well, once we adapted it to take account of GFP degradation. Our results allowed us to calculate values for the kdecay and GFPdecay parameters, something no-one had done before for our cell-free system.
Our cost modeling proved that our test was clinically viable and beneficial.
Visit our modeling page for more details.
