Meeting 1 ( 1300 to 1400)
The Detection System
- Fimbriae : Engineering a module within a nonpathogenic organism that allows for attachment to the target pathogen and subsequent triggering of responses.
- Get insight into physical structural basis of fimbriae ( contact Profs. Steve Mathews and Gad Frankel and The Institute of Hygiene and Tropical Medicine).
- One of the main concerns with this idea is that attachment to the host is one of the first responses in a host-parasite relationship which confers pathogenecity to the host leading to ethical concerns.
- Indirect detection : Look into excretion of potentially detectable molecules from the body surface of worms; other human worm parasites in addition to hook worms and schistosoma. Learn more about the environment, nutrition (anything that could be fed to the parasite that would alter it's metabolism and induce release of detectable substance) and life-cycle of the worms.
- Speak to Martha Betson who has worked with schistosoma in Uganda to gain further insight into detection.
- Take the idea further (by Tom) : further engineer a neutralization system which would have a detrimental effect on the parasite.
- Think about sensitivity : concentrations required for detection.
- (by James from Imperial 2009 iGEM team) Think about construction of a modular sensing component with variable receptor proteins. These could be comercially available antibodies. Binding of these antibodies to cell surface receptors initiate germination of bacterial spores. Extract the binding region of receptors that bind to a non-variable region of the antibody. (James is working on such a system involving fusion proteins in B. Subtillis. This sensing mechanism could then be coupled to a detection component. However, there are several concerns with this approach :
- Freely floating antibodies could trigger the sensing mechanism regardless of the actual parasites being within the sample tested - this could possibly be overcome by fc receprtors that express antibodies only when bound to the parasite.
- Antibodies are expensive and temperature dependant for storage.
- There is no real need for an intermediate sensing mechanism involving bacteria, the antibodies could be used directly.
- There are additional issues with concentrations (huge concs needed for detection), false positives and excretion.
The Response System
Main concern : SPEED; avoid transcription since this would probably be the rate determining step. Usually transcription takes between 30 and 60 minutes. Our response should ideally be faster than this.
- Fusion pigment protein / split pigment - final folded cofiguration of protein to produce a colour is usually complex with several overlapping regions. Formation of a split protein by cutting the pigment protein in half would cause the two halves to fold into unpredictable configurations which may not necessarily fold correctly to produce a colour upon interaction.
- FRET - maybe a better option because it does not rely on anzymatic catalysis to produce colour but rather on phosphorylation of a dimeric protein. This may also lead to faster response time.
- BRET - invloves the enzyme luciferase which is expensive to produce in the lab and would therefore not be an option for our application/s.
- Coloured pigments - involve extensive metabolic pathways for production of the colour. This increases the response time.
- Inteins (by Tom) - proteins that link two other proteins of interest and arecapableof splicing themselves out upon being triggered however this takes about an hour; again increasing response time.
- Proteases for cleavage - these need to be specfic such that they only cut at the linker sequence, non-toxic to the cell, produced within bacterial species of interest, fast and efficient and made of a small number of amino acids (in the case they need to be synthesized after detection)
General comments on the project
- Think about relevent bio-brick parts - can parts already in the registry be re-used or is it necessary to create them de novo making use of current literature?
- Waterbourne parasites in developing world is a huge problem. It is a foundational engineering problem that is technically solvable. However, it would need to be linked to a hugely impacting application.
Meeting 2 (1600 to 1700)
Skin proteases released by parasite
The parasite of interest, Schistosoma cercariae, produce proteases the digest skin lipids to trigger invasion. Thus skin lipids could be used to stimulate these proteases which could possibly be directly used to trigger two component systems that activate the effectors. The effector (a small peptide) usually tagged onto an extracellular receptor could then cleave a two component system and release a colour. Problems: the protease may still active but still fused to surface or the catalytic activity of the protease may be impaired by surface attachment. An alternative could be to use a soluble protease formed when two binding domains of proteins bring the fusion halves of the protease together. Problem : do they dimerize naturally without the need for activation. Dimerization is not an on off switch.