Hi Simon,
-- First of all, this might be interesting for you: http://ieeexplore.ieee.org/xpl/login.jsp?tp=&arnumber=1300016&url=http%3A%2F%2Fieeexplore.ieee.org%2Fxpls%2Fabs_all.jsp%3Farnumber%3D1300016
I'm currently at home so I could not download the full text, but the abstract sounds promising.
2015-02-03 19:32 GMT+01:00 Simon Rose <excaliburorama@googlemail.com>:
Hi Blenderkid and Gavin, sorry about the delay in responding, I've been offline for a few days !In response to Blenderkid's question, yes I intend to try to couple the TLR's to a cell. Initially I thought of using e.coli as a chassis, and as TLR2/6 respond to gram- positive bacteria and e.coli is gram negative, self- detection should not be a problem. It would be something on the lines of a little "machine" for sensing gram-positive pathogens. However, I am very unsure if the transmembrane domains of both TLRs are even capable of being anchored in the cell -wall of a gram negative bacterium, so I might have to use yeast as a chassis.
As for the display of the TLRs on the surface of E.coli, I'd recommend a fusion to ompA or any other kind of surface display:
http://www.sciencedirect.com/science/article/pii/S016777991000199X
http://www.sciencedirect.com/science/article/pii/S016777991000199X
Simply search for "E.coli surface display" for more information.
Also removing the transmembrane domain of a TLR does not obliterate its recognition ability per se, so using the entire extracellular region of the TLRs fused to ompA might be promising.
An example of TLR recognition sites (entire extracellular domain) used in fusion proteins: http://www.google.com/patents/EP1657257A1?cl=en
An example of TLR recognition sites (entire extracellular domain) used in fusion proteins: http://www.google.com/patents/EP1657257A1?cl=en
As for the problem with split GFPs flopping around and dimerising independently, well this should not be a problem if the TLR GFP constructs are anchored in a cell membrane/wall.
I would doubt that, as a membrane is somehow a fluid itself. Your protein will not be tightly bound on the membrane surface unless you attach it to the cytoskeleton. There will always be some background signal as a result of random dimerization, but hopefully it will be less than the positive signal when pathogens are present.
Anyway, one other concern might be the sensitivity of this approach, as the underlying signaling cascade, which amplifys the reaction to pathogen contact, is missing. You might end up with only a low fluorescence signal reducing the applicability of your method to wastely infested water puddles, instead of a blood samples for example.
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One other note for the time when you start testing your sensor: The samples that contain pathogens should be lysated prior to the detection experiments, as it virtually increases the amount of pathogen in your sample by dispersing the bacteria into tiny bits, instead of having intact bacteria in your solution. This might improve your signals.
Good luck with your project and please keep us up to date ;)
-Filip
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