Team:Thessaly/Demonstrate
DEMONSTRATE.
The main goal of our experiments was to develop a system that is not only functional, but can perform reliably under realistic conditions. We have developed a realistic method to isothermally amplify selected DNA biomarkers of disease agents. This method can be extrapolated to any disease and can detect target DNA in any form it exists in a biological sample. Detection is achieved through a linked, readily visibl, colorimetric assay, regulated by a toehold switch.
Can the system detect our biomarker?
We successfully amplified biomarkers from two different pathogenic agents, Mycobacterium tuberculosis (MTB) and Hepatitis B Virus (HBV). The amplification was successful both with conventional PCR, as well as with the isothermal amplification method RPA (Fig. 1). It is noteworthy that both methods were able to detect as little as 1 copy per 50μl reaction in an agarose gel. Furthermore, the addition of overhanging sequences to enable transcription and translation in downstream steps was also successful.
Figure 1. A) Detection of Mycobacterium tuberculosis biomarker IS6110 with Recombinase Polymerase Amplification (RPA). B) Detection of an HBV genome fragment using RPA.
Moreover, since Mycobacterium DNA, and hence our selected biomarker, is present as ~150bp fragments in a patient’s urine (the biological sample we chose for implementation of our project), we performed a random fragmentation assay to simulate the form of the MTB biomarker in urine (Fig. 2a) by treating template DNA with a human non-specific endonuclease. We were able to amplify the biomarker successfully even after near complete fragmentation of template DNA to lengths of ~100bp using DNAseI treatment (Fig. 2b).
Figure 2. A) Random fragmentation of the MTB IS6110 biomarker using DNaseI non-specific endonuclease. B) Amplification of the desired part of the DNaseI treated biomarker using PCR.
Can our engineered toehold-switch regulated system produce a colorimetric readout?
The next step in our project was the production of a highly efficient, sensitive and reliable colorimetric readout assay, utilizing an orthogonal toehold switch to regulate the translation of our selected reporter enzyme β-lactamase.
Figure 3. Enzymatic assay of β-lactamase with nitrocefin as its substrate, when expressed from a non-regulated and a toehold regulated construct in a cell-free system. Error bars correspond to standard deviation of n=2 replicates. Blank was subtracted.
Moreover, the sensitivity of the assay was high, generating a robust signal from trigger sequence concentrations as low as 7nM in a 1-hour assay.
Integrating two steps into one experiment
After verifying that the detection module and the colorimetric readout module work separately, we wanted to assess the performance of the two in an integrated system. The ultimate goal was to observe the behavior of the system when we used the output of the detection module as a trigger sequence input for the in vitro protein synthesis module (Fig. 4).
Figure 4. Measurement of toehold-regulated β-lactamase when the trigger is the previous step’s amplified sequence or in a plasmid vector. Error bars represent the standard deviation of n=2 replicates. Blank was substracted.
The activity of β-lactamase generated in this assay is similar when using a linear amplicon or the same sequence incorporated in a circular plasmid. Moreover, the signal produced in both cases is 5-fold higher than the no-trigger negative control. Thus, we verified that the system can produce a robust signal if we add the amplicon from the detection module as the trigger sequence. This result demonstrated the holistic functionality of our system.
Creating synthetic universal toehold switches
To further enhance the impact of our system in Synthetic Biology, we sought to create novel and universal parts for DNA detection. We designed an in silico pool of universal toehold switches, with trigger sequences deriving from hyperthermophile organisms. In this manner, we have designed unique trigger sequences that can reliably detect DNA sequences from any organism. To this end, we used these newly developed toehold switches in our project to detect an MTB DNA biomarker. Below we present the performance of the best toehold switch tested (Fig. 5).
Figure 5. Performance of toehold 13 in a β-lactamase enzymatic assay. Error bars represent standard deviation of n = 2 replicates. Blank was substracted.
As seen in Fig. 5, our universal toehold switch can reliably regulate the translation of β-lactamase mRNA, thus producing a robust signal in low trigger concentrations. However, due to lack of time we were only able to conduct one DBTL (Design - Build – Test – Learn) cycle. We hope that future efforts will improve on the part we designed, and thus enhance its sensitivity and specificity further.
In summary, we have successfully built a two-module system for the detection of MTB. These two modules allow for the detection of disease biomarkers and for generating a readout based upon detection function orthogonally. Moreover, we showed that this system is highly sensitive and that it is readily adaptable for the detection of any disease agent’s DNA.
Is our project simple to implement?
The final implementation of our project is in refugee camps and limited resource settings, underlining the need for a simple and safe diagnostic test. For this reason, we tried to transform the complexity of our lab’s experimental steps into a product incorporating the feedback from our stakeholders’ interactions. We begun by designing our prototype and having it 3D printed and then we ran a simulation of its usage at a Refugee Accommodation Facility. During the simulation, we confirmed that is simple and easy to implement in such a facility. We also pointed out that our prototype does not bear all the features of its final implementation, as it was developed during limited time.
Figure 6. Our 3D printed prototype
