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Team:KULeuven/Concreties
From 2008.igem.org
Op deze pagina verzamelen we concrete invullingen omtrent het project. De bedoeling is niet meer om over conceptuele dingen na te denken, maar om bijvoorbeeld op zoek te gaan naar analogen met vorige projecten, bruikbare BioBricks enzoverder. Voorlopig gooien we het nog niet onmiddellijk op de voorgrond, tot we meer voet in de aarde hebben omtrent haalbaarheid en zo.
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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)
- The input signal is of course arabinose (we need AraBAD 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”.
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.
- 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?
- If cAMP is not degraded fast enough, could cyclic nucleotide phosphodiesterases catalyse this process of degradation?
