Proposal & Approach
From 2006.igem.org
| Backround and Signalling Pathway | Proposal & Approach | Anticipated Results & Significance | Troubleshooting, References & Sponsors | Team members |
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Project proposal
In the beginning we have discussed following project ideas:
- New mechanism of tolerance - use of inhibitors that interfere with NF-kappaB (transcription factor mentioned above) - inhibition with dominant negative proteins involved in signaling pathway, this proteins could be labeled with degradation tags (PEST sequence) and inhibition would be temporal (negative feedback loop)
- Cell response to pathogen, that cells usualy can not recognise - for example response to beta glucans of fungi
- Find a connection/shortening of signaling pathways to make it more efficient and include more responses
Selected project proposal
The basic idea of our project was to introduce the feedback loop,which would decrease the response to the persistent or repeated stimulus. However completly shutting down the response at bacterial stimulation is not a good solution. Ideally the feedback loop should decrease the response when it is too high but recover the responsiveness of the system after some time. Inhibition of the response could be achieved by activating the expression of the dominant-negative adapter protein, that inactivates the signaling pathway. Decreasing the lifetime of the dominant-negative inhibitior by the addition of rapid degradation tag (PEST sequence) should inactivate the inhibition and reset (restore) the normal responsiveness of the immune system.
This idea is similar to the natural mechanism of tolerance, which is already present in living cells and which decrease the response to repeated bacterial stimulation. This natural tolerance is activated slowly, on the order of days and operates through several different mechanisms (Figure). Our feedback mechanism (i.e. artificial tolerance) should decrease the response within hours and thus "attack" the signaling pathway at the point, which has not been used in the natural system.
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