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Talk:Team:KULeuven/Concreties
From 2008.igem.org
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Oude ideeën
Input
Andere mogelijkheden
- There is a BioBrick (BBa_I765001) that is induced by UV-light. This would be perfect: only UV light could than trigger our system. This BioBrick is not confirmed to work, and there is very little information availabla about it. I doubt its proper working, however.
- Harvard 2005 also did something with UV. It involved the lambda cI repressor, and resulted in UV light switching something on. I do not (yet) understand how it works. See http://openwetware.org/images/8/86/Doucette_et_al._2006.pdf.
Output
Filter
Idea 1
This is what I found (up to now).
- In the paper of Shen-Orr , the example of the feedforward loop with AND-gate is the well-studied Ara-operon (This can occur when X and Y act in an ‘AND-gate’−like manner to control operon Z, as is the case in the araBAD operon in the arabinose feedforward loop)
- Is dit het enige voorbeeld dat ze aanhalen in shen-orr? misschien is dit systeem nogal moeilijk omvormbaar naar een specifieke filter vermits cAMP nogal een belangrijke rol speelt in de cel – het idee was dan ook om de input van X te veranderen in een induceerbare promoter naar keuze, bijv een OmpR afhankelijke als we het systeen van levskaya met de lichtpuls gebruiken -- IT
- The input signal is of course arabinose. We will need AraF(GH?) to transport it efficiently from the medium into the cell.
- What is depicted (see figure) as X, is cAMP (+ CAP)
- Y is AraC
- The AND gate is the “unlooping” of the DNA strand in de Ara operon. This happens only in the presence of both AraC bound to arabinose, and CAP-cAMP
- Product Z are in a WT Ara operon the AraBAD proteïns, but can of course be changed to eg. lactonase. The araBAD genes are located downstream the pBAD promoter, which is activated by the “DNA unlooping”.
- Kan Z ook een tussenproduct zijn dat enerzijds de inverter ingaat richting HSL productie en anderzijds, na het ribolock-key systeem, lactonase productie aanstuurt -- IT
Note that Y is also to be stimulated by arabinose, so the scheme is actually a bit more complex than the easy scheme depicted in the Shen-Orr paper
Some problems
- I cannot find literature about the dotted purple arrow: is it überhaupt existing? The article of Shen-Orr suggests it does.
- cAMP concentration is depressed by glucose, so maybe the purple arrow means that the concentration of glucose is negligible compared to arabinose. This might however not be good enough.
- What could be done, is making sure that not only AraBAD is made upon (eg) flashing with a light (Austin iGEM 2004), but also making a repressor for the phosphotransferase system of glucose transport. However, then, the pink purple arrow should be the full (non-dotted) pink one.
- I did not (yet) find the BioBricks of the Austin 2004 team for the input signal.
- I believe they can be found here: http://partsregistry.org/Featured_Parts:Light_Sensor -- IT
- Adenylate cyclase inhibition is coupled to the flux of glucose through the glucose PEP:phosphotransferase system . This means that external glucose concentration regulates cAMP production. This is I think a major problem: how do we arrange the system so that it becomes dependent on cytoplasmatic factors?
- Vandaar dat ik hoop dat er nog een ander systeem in E.coli gekend is dat dit netwerkmotief heeft, daar waren we een beetje vanuit gegaan... -- IT
- Ik denk dat het systeem zoals hierboven uitgelegd moet lukken. Prof. Winderickx vertelde dat cAMP zowel afhankelijk is van intra- als extracellulaire glucose-concentratie. Dat lost het probleem hier geformuleerd op. Daarenboven klinkt de paper die hieronder geciteerd staat zeer veelbelovend, zij hebben immers al uitvoerig het ara-operon in min of meer hetzelfde perspectief als hier beschreven bestudeerd. Bmoeyaert
- If cAMP is not degraded fast enough, could cyclic nucleotide phosphodiesterases catalyse this process of degradation?
Interesting reads
Idea 2
- schema filter 1
- Is probably not working, because the T7 polymerase (even with tag) is much more stable than the RNA Bmoeyaert
