Simon writes:
> Hi everyone I'm interested in making a synthetic pathogen sensor using human toll- like receptors 2 and 6.
Wikipedia and [2][3] indicate that these operate by "recogniz[ing] the gross, primarily structural features of molecules not innate to the host organism[1]". So really the resulting "OMG! IT'S A PATHOGEN!" signal is probably the product of two things: the fact that the receptor recognized the molecule AND the fact that the molecule was actually there where the receptor was looking for it. So inside a human it works somewhat like a guard patrolling a locked warehouse at night, who doesn't have to be terribly discriminating as pretty much anyone they run into at 2am has a high likelihood of being an intruder. But if you put that guard on a crowded city street, the fact that they're suddenly seeing lots of unknown people is not terribly interesting. So I wonder if the proposed system would be specific enough to be useful outside of the biological environment it evolved to serve.
You might have a hard time finding a procaryote to express this system in (if I correctly understand that to be your goal) that isn't itself something that the system will recognize as a "pathogen". [2] indicates that TLR2 for example is sensitive to components of gram-positive bacteria and yeasts. And if you express it in a gram-negative organism like E. coli, does the more complicated cell-envelope mean that it's hard to get the recognition end of the protein sufficiently "outside" for it to encounter the things it's looking for? But looking at [2] again another TLR2 substrate is triacylated lipoprotiens which I think you'll find in E. coli too.
> The idea is to ligate the pathogen-sensing, extracellular domains of each TLR (or possibly just the recognition module,
> consisting of just a single residue) to one half of a GFP, producing a visible signal when the TLRs dimerise.
Today I Learned that "In split GFP, two fusion proteins are produced, each one is fused to "half" of a GFP protein (its not exactly half but let not go into that now). If the two fused proteins are in close proximity, the two halves associate to produce an GFP that fluoresce irreversibly[4]." Neat.
However I think this may not help in your application because while the dimerization of the TLRs in the presence of the things they're looking for may bring the split GFPs together, there's probably nothing stopping them from from finding each other without the TLR dimerization, so you may end up with a bunch of TLRs flopping around while connected by their linked GFPs, and lots of florescence under all circumstances.
> Using a prokaryotic chassis to carry the TLRs might not be possible owing to the absence of endoplasmic reticula in prokaryotes,
> and the consequent problems with protein folding, as well as with being able to anchor the transmembrane domains of the TLRs
> in the cell wall. Any TLRs expressed might not fold properly.
Also potentially the procaryote's collection of lipid species in the membrane might not be what it's expecting (compare the simplicity of the E. coli cell membrane lipid composition to that of a human cell (no cholesterol in E. coli (IIRC) for example)) so even if it folds reasonably well it still might not function perfectly (but for your application I guess you don't care). And somehow all of the recognition end of the thing is going to have to get through (at least) the plasma membrane along with this GFP that's stuck to it.
> I think that TLR signal transduction is not well understood hence the use of GFPs as reporters, as bacteria would lack the requisite
> intracellular signalling pathways. If these problems prove insuperable, then a eukaryotic chassis might have to be used.
You could always do Drosophila, which already include TLRs and have the advantage of providing a self-propelled mobile aerial sensor platform. There you just need to connect some downstream part of the
signaling cascade with a GFP reporter and watch out for glowing flies (something, something, automated florescence detector, something, something, banana).
> Anyway, it's a learning curve!
Good luck with it!
[1]http://en.wikipedia.org/wiki/Toll-like_receptor
[2]http://www.sciencedirect.com/science/article/pii/S0969212611000724
[3]http://www.jleukbio.org/content/70/4/467/F3.expansion.html
[4]https://greenfluorescentblog.wordpress.com/tag/split-gfp/
[5]http://www.ncbi.nlm.nih.gov/gene/7097
-- > Hi everyone I'm interested in making a synthetic pathogen sensor using human toll- like receptors 2 and 6.
Wikipedia and [2][3] indicate that these operate by "recogniz[ing] the gross, primarily structural features of molecules not innate to the host organism[1]". So really the resulting "OMG! IT'S A PATHOGEN!" signal is probably the product of two things: the fact that the receptor recognized the molecule AND the fact that the molecule was actually there where the receptor was looking for it. So inside a human it works somewhat like a guard patrolling a locked warehouse at night, who doesn't have to be terribly discriminating as pretty much anyone they run into at 2am has a high likelihood of being an intruder. But if you put that guard on a crowded city street, the fact that they're suddenly seeing lots of unknown people is not terribly interesting. So I wonder if the proposed system would be specific enough to be useful outside of the biological environment it evolved to serve.
You might have a hard time finding a procaryote to express this system in (if I correctly understand that to be your goal) that isn't itself something that the system will recognize as a "pathogen". [2] indicates that TLR2 for example is sensitive to components of gram-positive bacteria and yeasts. And if you express it in a gram-negative organism like E. coli, does the more complicated cell-envelope mean that it's hard to get the recognition end of the protein sufficiently "outside" for it to encounter the things it's looking for? But looking at [2] again another TLR2 substrate is triacylated lipoprotiens which I think you'll find in E. coli too.
> The idea is to ligate the pathogen-sensing, extracellular domains of each TLR (or possibly just the recognition module,
> consisting of just a single residue) to one half of a GFP, producing a visible signal when the TLRs dimerise.
Today I Learned that "In split GFP, two fusion proteins are produced, each one is fused to "half" of a GFP protein (its not exactly half but let not go into that now). If the two fused proteins are in close proximity, the two halves associate to produce an GFP that fluoresce irreversibly[4]." Neat.
However I think this may not help in your application because while the dimerization of the TLRs in the presence of the things they're looking for may bring the split GFPs together, there's probably nothing stopping them from from finding each other without the TLR dimerization, so you may end up with a bunch of TLRs flopping around while connected by their linked GFPs, and lots of florescence under all circumstances.
> Using a prokaryotic chassis to carry the TLRs might not be possible owing to the absence of endoplasmic reticula in prokaryotes,
> and the consequent problems with protein folding, as well as with being able to anchor the transmembrane domains of the TLRs
> in the cell wall. Any TLRs expressed might not fold properly.
Also potentially the procaryote's collection of lipid species in the membrane might not be what it's expecting (compare the simplicity of the E. coli cell membrane lipid composition to that of a human cell (no cholesterol in E. coli (IIRC) for example)) so even if it folds reasonably well it still might not function perfectly (but for your application I guess you don't care). And somehow all of the recognition end of the thing is going to have to get through (at least) the plasma membrane along with this GFP that's stuck to it.
> I think that TLR signal transduction is not well understood hence the use of GFPs as reporters, as bacteria would lack the requisite
> intracellular signalling pathways. If these problems prove insuperable, then a eukaryotic chassis might have to be used.
You could always do Drosophila, which already include TLRs and have the advantage of providing a self-propelled mobile aerial sensor platform. There you just need to connect some downstream part of the
signaling cascade with a GFP reporter and watch out for glowing flies (something, something, automated florescence detector, something, something, banana).
> Anyway, it's a learning curve!
Good luck with it!
[1]http://en.wikipedia.org/
[2]http://www.sciencedirect.
[3]http://www.jleukbio.org/
[4]https://
[5]http://www.ncbi.nlm.nih.
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