Team:Athens/Description
FOR THE FULL EXPERIENCE PLEASE USE A PC :)
Description
Inspiration
2020 was a year of sudden changes. Among others, the urgent need for surveillance, prevention, preparedness, and control of a pandemic-prone disease resulted in an exponential worldwide rise in demand for the detection of a new emerging pathogen.
The PCR technique has been, over the years, a necessary tool for research and is considered by many the gold standard of molecular diagnostics. However, in the context of this past year’s large-scale testing, the RT-PCR procedure has indicated a clear problem regarding the availability, affordability, and accessibility of its raw materials. According to data from September 2020, Europe experienced significant differences in the testing rate across its population. Back then the likelihood of being tested could differ up to 30 times between the EU Member States, which would, in turn, influence the test positivity rate.
Identifying the problem
PCR protocol requires a mixture of deoxynucleoside triphosphates (dNTPs), or simply nucleotides - the individual building blocks of DNA - and a DNA polymerase, which are the major components of the PCR Master Mix. The production of dNTPs depends on a centralized industrial market, their total cost is high and they require refrigeration at -20oC. These restrictions hamper the prompt and efficient response of diagnostic centers that make extensive use of the PCR technique for testing.
The industrial production of dNTPs is still based - to a certain extent - on non-standardized, low-yield, unsustainable chemical processes. Chemically synthesized dNTPs depend on separate pipelines and require proper handling performed by skilled staff including isolation, purification, storage, and recycling of environmentally hazardous waste products.
Zooming out
The above technical difficulties also affect the application of other nucleic acid amplification (NAA) techniques as well, such as microfluidics-based PCR devices. Since these sample-to-answer systems are mainly applied in point-of-care applications, there is an urgent need for more equitably distributable, easy-to-use, and low-cost reagents for such diagnostic tests in remote settings.
Connecting puzzle pieces
Designing an enzymatic system that can sustainably and automatically produce dNTPs and DNA polymerase in a streamlined way using affordable substrates is a basic step towards a widely accessible PCR and thus a more substantial emergency preparedness and response.
Project Goal
Considering all the mentioned aspects of both the PCR technique requirements and the difficulties in the dNTPs' production, we created the project AdAPTED. With our project we propose a novel biological system that can produce, de novo, dNTPs, as well as Pfu DNA polymerase. The key point of our project is based on taking advantage of the natural ways organisms produce, de novo, dNTPs, as it is essential for their reproduction states. This is possible due to enzymatic reactions that include the Ribonucleotide Reductase (RNR) and Thymidylate Synthase (TSase) enzymes. The synergetic action of both enzymes results in the conversion of ribonucleotides triphosphates (NTPs) to dNTPs which are essential for the production of DNA. These enzymes control the rate of the production of dNTPs as other metabolic pathways needed are abundant to the cell. By using different promoters for the regulation of these enzymes, elevated dNTPs production is ensured.
Based on this knowledge, we plan to create two distinct genetic circuits. One that will overexpress RNR and TSase, in order to significantly increase the dNTP pools in the cell, and a second one that produces Pfu DNA polymerase. Both reagents, dNTPs and Pfu polymerase, could be provided to the end-user through three different scenarios:
- as a lyophilized recombinant plasmid to be followed by transformation of E. coli strains, that will produce the desired reagents in the point of need
- as part of the provided lyophilized cell lysate
- as isolated and purified reagents
In addition, experiments will be conducted to measure the overexpression levels needed for RNR and TSase, the time needed to reach adequate dNTPs concentrations and possibly the automation of the process with a hardware.
The selected organism for these experiments is the E. Coli strain BL21. This strain was selected due to its ability to overexpress enzymes, with the correct promoters, and has also been shown to produce Pfu DNA polymerase. Regarding the DNA polymerase, it was evident to us that it is a big part of the materials costs and required as much handling as dNTPs. So aiming to achieve a “one-pot” PCR level of accessibility, we added the production of Pfu polymerase to the proposed biological system. Pfu polymerase was selected due to its low-error rate and wide use in diagnostics and molecular biology applications and will be N-His tagged for an easy extraction.
The proposed enzymatic synthesis of dNTPs uses low cost raw materials, is sustainable and has the potential to be automated. The final product is designed to be either the open outsource of the plasmid, for labs to produce the reagents based on their needs, or a lyophilized lysate that contains the PCR reagents and can be stored in room temperature, based on the needs of each end user. Lastly, this system can be acquired and used by labs around the world and can also be upscaled to provide an industrial sustainable way of production of these reagents. Through our project we hope to facilitate the standardization and accessibility of PCR technique, in a way that meets the constant rise in demand of diagnostic testing.
Our vision
dNTPs and DNA polymerase do present major restrictions in terms of accessibility, affordability, complexity and sustainability, upon which we built our brainstorming. Our project draws inspiration from all these parameters that hamper diagnostic centers’ prompt and efficient response, especially when needed the most: during crisis management. Through the design of an enzymatic system that can directly, easily and sustainably produce dNTPs and DNA polymerase in a streamlined way using affordable substrates, we have a double vision: to encourage the industrial production shift of raw materials used in diagnostics to simpler and greener alternatives and at the same time facilitate their decentralization and application in remote communities worldwide. Through this bottom-up approach, we aim to set requirements for universally accessible diagnostic tests and thus a more substantial future emergency mindfulness. We aim to achieve this by opening the way for a functional up-scaled biological system that can competently replace the current production of dNTPs and DNA polymerase in industrial settings, while also making choices that enable its wide distribution and use.
1. Hasell, J., Mathieu, E., Beltekian, D., Macdonald, B., Giattino, C., Ortiz-Ospina, E., Roser, M., & Ritchie, H. (2020). A cross-country database of COVID-19 testing. Scientific Data, 7(1). https://doi.org/10.1038/s41597-020-00688-8
2. Rannan-Eliya, R. P., Wijemunige, N., Gunawardana, J., Amarasinghe, S. N., Sivagnanam, I., Fonseka, S., Kapuge, Y., & Sigera, C. P. (2020). Increased Intensity Of PCR Testing Reduced COVID-19 Transmission Within Countries During The First Pandemic Wave. Health Affairs, 10.1377/hlthaff. https://doi.org/10.1377/hlthaff.2020.01409
3. Mercer, T. R., & Salit, M. (2021). Testing at scale during the COVID-19 pandemic. Nature Reviews Genetics, 22(7), 415–426. https://doi.org/10.1038/s41576-021-00360-w
4. Vandenberg, O., Martiny, D., Rochas, O., van Belkum, A., & Kozlakidis, Z. (2020). Considerations for diagnostic COVID-19 tests. Nature Reviews Microbiology. https://doi.org/10.1038/s41579-020-00461-z
5. Larremore, D. B., Wilder, B., Lester, E., Shehata, S., Burke, J. M., Hay, J. A., Tambe, M., Mina, M. J., & Parker, R. (2020). Test sensitivity is secondary to frequency and turnaround time for COVID-19 screening. Science Advances, eabd5393. https://doi.org/10.1126/sciadv.abd5393
6. EU health preparedness: Recommendations for a common EU testing approach for COVID-19, Agreed by the Health Security Committee on 17 September 2020, EUROPEAN COMMISSION DIRECTORATE-GENERAL FOR HEALTH AND FOOD SAFETY.
7. Population-wide testing of SARS-CoV-2: country experiences and potential approaches in the EU/EEA and the United Kingdom, European Centre for Disease Prevention and Control, 19 August 2020.
8. Strategies for the surveillance of COVID-19, European Centre for Disease Prevention and Control, 2020.
9. Diagnostic testing and screening for SARS-CoV-2, European Centre for Disease Prevention and Control, 8 September 2021.
10. Guan, D., Wang, D., Hallegatte, S., Davis, S. J., Huo, J., Li, S., Bai, Y., Lei, T., Xue, Q., Coffman, D., Cheng, D., Chen, P., Liang, X., Xu, B., Lu, X., Wang, S., Hubacek, K., & Gong, P. (2020). Global supply-chain effects of COVID-19 control measures. Nature Human Behaviour, 4(6), 577–587. https://doi.org/10.1038/s41562-020-0896-8
11. Zhu, H., Zhang, H., Xu, Y., Laššáková, S., Korabečná, M., & Neužil, P. (2020). PCR past, present and future. Biotechniques. https://doi.org/10.2144/btn-2020-0057
12. Infographic vector created by freepik - www.freepik.com