Difference between revisions of "Team:Oxford/Description"
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<p>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 was produced in response to the presence of cruzipain, the blood would not clot. The 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.</p> | <p>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 was produced in response to the presence of cruzipain, the blood would not clot. The 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.</p> | ||
<p>For the 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 will be produced. In the protein based system, both our input (cruzipain) and our intermediate output (TEV protease) are proteases. We introduced an amplification step to improve the final output (active bivalirudin) of our circuit – the produced TEV protease will cleave A–B* linkers, thus increasing levels of TEV protease and inactive bivalirudin, to activate it. As a result, our system will require only a small initial signal to produce a clear response.</p> | <p>For the 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 will be produced. In the protein based system, both our input (cruzipain) and our intermediate output (TEV protease) are proteases. We introduced an amplification step to improve the final output (active bivalirudin) of our circuit – the produced TEV protease will cleave A–B* linkers, thus increasing levels of TEV protease and inactive bivalirudin, to activate it. As a result, our system will require only a small initial signal to produce a clear response.</p> | ||
| − | <p>Our second system is a protein circuit encased in outer membrane vesicles (OMVs). OMVs are constitutively produced by Gram-negative bacterial cells and contain periplasmic solution. We targeted the proteins to outer membrane vesicles with OmpA and SpyTag/SpyCatcher. One of the main components of our system is a split TEV protease, whose two halves are made accessible to each other in the presence of cruzipain – the protease is activated upon dimerization and can go on to activate more of itself in an amplificatory manner. Active TEV protease can then cleave and release bivalirudin, which acts as the reporter of our system by inhibiting blood clotting.</p> | + | <p>Our second system is a protein circuit encased in outer membrane vesicles (OMVs). OMVs are constitutively produced by Gram-negative bacterial cells and contain periplasmic solution. We targeted the proteins to outer membrane vesicles with OmpA and SpyTag/SpyCatcher. One of the main components of our system is a split TEV protease, whose two halves are made accessible to each other in the presence of cruzipain – the protease is activated upon dimerization and can go on to activate more of itself in an amplificatory manner. Active TEV protease can then cleave and release bivalirudin, which acts as the <a href = "https://2017.igem.org/Team:Oxford/Design">reporter</a> of our system by inhibiting blood clotting.</p> |
<--! insert project design page logo & link to right of text --> | <--! insert project design page logo & link to right of text --> | ||
Revision as of 21:35, 31 October 2017
Project Description
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 (possible through synbio) allows for multiple diagnostic variables to be processed in a dynamic fashion, aiding precise health care decisions and 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?
We identified a gap in the field of rapid, point-of-care diagnostics which arises when antibody-based technologies cannot be used, e.g. 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
<--! insert Chagas page logo -->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 was produced in response to the presence of cruzipain, the blood would not clot. The 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 the 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 will be produced. In the protein based system, both our input (cruzipain) and our intermediate output (TEV protease) are proteases. We introduced an amplification step to improve the final output (active bivalirudin) of our circuit – the produced TEV protease will cleave A–B* linkers, thus increasing levels of TEV protease and inactive bivalirudin, to activate it. As a result, our system will require only a small initial signal to produce a clear response.
Our second system is a protein circuit encased in outer membrane vesicles (OMVs). OMVs are constitutively produced by Gram-negative bacterial cells and contain periplasmic solution. We targeted the proteins to outer membrane vesicles with OmpA and SpyTag/SpyCatcher. One of the main components of our system is a split TEV protease, whose two halves are made accessible to each other in the presence of cruzipain – the protease is activated upon dimerization and can go on to activate more of itself in an amplificatory manner. Active TEV protease can then cleave and release bivalirudin, which acts as the reporter of our system by inhibiting blood clotting.
<--! insert project design page logo & link to right of text -->