Re: [DIYbio] TLR Biosensor

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] suggest 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

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