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.
The TMs of TLRs 2 and 6 are both 21 AAs in length by the way. There is some information about them on PLOS1. Apparently isolated TMDs are capable of oligermerisation independently of any PAMPS detected by the extracellular portions (PLOS 1 November 2012 | Volume 7 | Issue 11 | e48875 )
As for detection one idea would be to extract the TLR from say, HeLa ( assuming they're not too badly mutated) having tagged the primers with histidine codons, ligate into an expression vector and then detect any expressed protein with anti-6His antibodies, just to see if anything happens.
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. There has actually been some fairly recent work on fluorescent biosensors here;
Eiji Nakata, FongFong Liew, Shun Nakano and Takashi Morii (2011). Recent progress in the construction methodology of fluorescent biosensors based on biomolecules, Biosensors - Emerging Materials and Applications, Prof. Pier Andrea Serra (Ed.), ISBN: 978-953-307-328-6,rey ss b (
These ideas are still in progress by the way.
Its nice to have an international community to help out. The immune system fascinates me, by the way which is why I'm doing this! Thanks for the responses so far!
On Saturday, January 31, 2015 at 7:52:54 PM UTC, Simon Rose wrote:
Hi everyone I'm interested in making a synthetic pathogen sensor using human toll- like receptors 2 and 6 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. 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. 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. Anyway, it's a learning curve!
On Saturday, January 31, 2015 at 7:52:54 PM UTC, Simon Rose wrote:
Hi everyone I'm interested in making a synthetic pathogen sensor using human toll- like receptors 2 and 6 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. 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. 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. Anyway, it's a learning curve!
On Saturday, January 31, 2015 at 7:52:54 PM UTC, Simon Rose wrote:
Hi everyone I'm interested in making a synthetic pathogen sensor using human toll- like receptors 2 and 6 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. 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. 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. Anyway, it's a learning curve!
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