Difference between revisions of "Team:Oxford/Applied Design"

Why are synthetic biology diagnostics useful?

Conventional diagnostics are currently limited by factors such as resource availability and cost. Synthetic biology provides an opportunity for existing sophisticated biological designs to be exploited and integrated into new systems. Multiplexed signal processing allows for dynamic processing of multiple diagnostic variables, aiding precise health care decisions therefore directly benefiting doctors and patients. Importantly, this form of biotechnology is far more cost-effective and can support developing areas with poorer infrastructure. We therefore believe that synthetic biology diagnostics lie at the heart of the future of medicine.

Why did we focus on diagnostics?

Very early on, we each came up with an idea for our iGEM project and presented it to the group. You can see some of these on our Initial Ideas page.

We carried out a public survey in the UK, where more than half of the 200 surveyed wanted a synthetic biology solution for disease diagnosis. You can read more about our surveys on our Silver Human Practices page.

We identified a gap in the field of rapid, point-of-care diagnostics which arises when antibody-based technologies cannot be used, for example diagnosis of diseases in infants or immunocompromised patients. As a result, we decided to use the flexibility and versatility of synthetic biology to design a platform technology which addresses these issues.

What is Chagas disease?

Our cell-free diagnosis kit is designed to diagnose Chagas disease in its acute phase using a simple blood test. Chagas disease is a neglected tropical disease endemic to Latin America that impacts 6-7 million people, of whom 95% lack sufficient diagnosis or treatment. We decided to focus our efforts on designing a diagnostic for congenital Chagas disease, since current point-of-care diagnostics cannot be used to detect Chagas disease in infants. Current treatments using benznidazole and nifurtimox are almost 100% effective if given shortly after the onset of the acute phase. However, lack of diagnosis leads to the onset of the chronic phase, which causes irreversible pathological consequences to the heart, digestive system, and nervous system. We hope to make a positive contribution towards this cause with our project.

You can read more about this on our Chagas disease page.

What is our solution?

We have designed two systems - one DNA based and one protein-based - to detect a protease, cruzipain. Cruzipain is produced and secreted by T. cruzi in the blood and has a specific cleavage sequence, which is ideal for the input. Our systems have bivalirudin as the output for both methods. Bivalirudin is a small peptide that acts as an anticoagulant. Therefore if bivalrirudin were produced in response to the presence of cruzipain, the blood would be inhibited from clotting. These systems are designed to be cell-free and freeze-dried to ensure safety and ease of transport, before being added to a sample of blood.

For our DNA-based system, we have designed a TetR molecule with a cleavage site for TEV protease. Our TetR will start bound to its DNA operator, repressing the production of an output protein. When it is cleaved by TEV, repression is relieved, and the reporter produced.

For our protein-based system, we have designed an amplificatory protein circuit encased in outer membrane vesicles (OMVs). Both our input (cruzipain) and our intermediate output (TEV protease) are proteases. The amplification components of our system is a split TEV protease, the two halves of which are made accessible to dimerise in the presence of cruzipain. Upon dimerisation, the protease is activated and can go on to activate more of itself in an amplificatory positive feedback loop. Active TEV protease can then cleave and release bivalirudin, which acts as the reporter of our system by inhibiting blood clotting.

You can read more about this on our Design page.

What is our strategy?

Wet Lab

For our DNA-based system, we characterised the pTet + eYFP part using fluorescence microscopy and plate reading, which showed that TetR can bind to the pTet and repress the output fluorescence significantly. This part has a carefully picked ribosome binding site and promoter strength to optimise our system for minimal false positives and negatives when eYFP is replaced with TEV protease production. Hence it was highly important to detect how repression was relieved when Anydrotetracyline(ATC) was introduced, which acts on TetR.

The sfGFP+Quencher was characterised for our OMV system. This part was critical to identify if sfGFP (GFP modified to fold in the periplasm) can be quenched by a quenching peptide linked with a protease specific cleavage sequence. We tested the functionality and sensitivity of the part to TEV protease through a double transformation of the part and TEV plasmids. Plate reader and fluorescence microscopy on this part identified that the Quencher can quench sfGFP fluorescence and that quenching can be relieved by introducing the TEV protease.

You can read more about this in our Wet Lab section.

Real-world perspectives

Our project has been guided throughout by input from experts in Latin America and medical professionals in the UK. Conversations with the public during our outreach activities also helped us to consider perspectives around synthetic biology outside the lab. You can read more about this on our Gold & Integrated Human Practices page and Education & Public Engagement page.

Consultation with relevant stakeholders, including HeLEX (Centre for Health, Law and Emerging Technologies), InSIS (Institute for Science Innovation and Society) and numerous experts worldwide, has helped to inform ethical and social considerations relevant to our project. These consultations have directly fed back into our applied design to enable a bedside-to-bench approach helping us to design and prototype a diagnostic kit for Chagas disease which is easy-to-use, cheap to manufacture and has minimal risk to the environment. You can read more about this on our Applied Design page.

To support the integration of our device into existing healthcare systems, our dialogue with HeLEX inspired us to create a policy proposal to address gaps in regulation present in current infrastructure.

Our cell-free design has been inspired by consultation with Dr Keith Pardee. Combining this with our discussions about safety with Piers Millet and HeLEX, we designed our parts for the wet lab with this in mind and produced a report outlining the barriers faced by cell-free technology. We hope this will prove useful for future iGEM teams using cell-free technology.

Modelling

Modelling was an inseparable part of our design process: it allowed us to quickly test our theoretical designs and identify key design parameters that could improve our design. We worked closely with experts throughout developing our models. Collaborations have allowed us to refine our methodology by applying it to the different systems of other teams, inspiring us to document it to help future teams. You can read more about this in our Modelling section.

We were able to model the impact our diagnostic would have on the epidemiology of Chagas disease in Bolivia by working closely with Professor Michael Bonsall (a mathematical biologist) and Dr Yves Carlier (a Chagas epidemiologist) to create a disease model that we hope to publish later this year. You can read more about this on our Disease Modelling page.

What are our visions for the future?

Experiments we want to carry out

To develop our system into something which can undergo clinical trials and hopefully become a successful product, we have a vision for the experiments that need to be performed. These are detailed at the end of our Results pages - DNA-based and Protein-based.

For our DNA-based system, we envision the progression from a proof-of-concept system to gradually introducing each ‘real’ components, and testing that this does not perturb our system and corroborates our modelling. Additionally, we wish to check the efficacy of different lysates and the freeze-drying process.

For our protein-based system, the aim is to first express our components in outer membrane vesicles, before trialing methods of lysing the OMVs and assaying the efficacy of the split-TEV protease molecule.

Future visions for our kit

We have designed a software tool to facilitate further applications of our project, as our system may be applied to a range of diseases. This is an open-source tool so that researchers may add to a growing database of pathogens and specific protease cleavage sites.

As our kit is modular, it can be easily and cheaply adapted to diagnose different diseases: the cost of changing the disease is then only the input block, not also the output block. Our vision for the future is that a streamlined manufacturing process can be established for rapid development of new diagnostic modules as more specific proteases are characterised and validated as biomarkers.

References

Courbet A., Renard E., and Molina F. 2016 Bringing next‐generation diagnostics to the clinic through synthetic biology. EMBO Mol Med 8: 987–991

Slomovic S., Pardee K., and Collins J.J. 2015 Synthetic biology devices for in vitro and in vivo diagnostics. Proc Natl Acad Sci USA 112: 14429–14435.

Wehr, M. C. et al. 2006 ‘Monitoring regulated protein-protein interactions using split TEV’, Nat Meth, 3(12), pp. 985–993. Available at: http://dx.doi.org/10.1038/nmeth967.

Alves, N. J. et al. 2016 ‘Protecting enzymatic function through directed packaging into bacterial outer membrane vesicles’, Scientific Reports. Nature Publishing Group, 6(1), p. 24866. doi: 10.1038/srep24866.