Fv-fragment guided input module
To design detection-input mechanism with modular character, as opposed to the use of possibly existing two-component signalling cascades with specificity to e.g. parasite larval stages, we could exploit the specificity of antibodies to their substrate. Several teams in iGEM have used Ab's, attempting to alter the specificity of cell-surface receptors towards a desired target (e.g. Illinois 2008). To circumvent problems with direct manipulation of the receptor molecule, while incorporating the wide spectrum of potential targets, that could be recognized in an anitbody based approach, the Fv fragment of a given antibody is fused to the ligand of a suitable native two-component system. Figure 2 illustrates the principle of the Fv-fragment guided detection system.
- Expression of the Fv-ligand-fusion protein (detector protein). Since the production of Fv fragments in prokaryotes has not been successful so far, a eukaryotic system is preferred (e.g. Yeast).
- The detector protein is secreted into the extracellular environment.
- Due to the high specificity of the Fv-fragment to its substrate, the detector protein binds to a given pathogen.
- Spatial co-localization of detector proteins on the pathogens surface is mirrored by clustering of receptors for the ligand-fragment, in the detector cell. This leads to the induction of a downstream signalling cascade to trigger a response module.
The system has several features which render it interesting:
- An extremely broad array of potential targets that can be detected, exploiting the power of molecular recognition via Antibody fragments.
- A highly modular character. Exchangebility of specificity and potential to link the system to virtually any given response system
- Very close to real world applications (parasites, toxins etc).
Two central issues come along with this system:
- A suitable secretion system has to be found to allow efficient trafficing of the detector protein into the extracellular space.
- Production of false positives has to be prevented. A requirement for clustering and crosslinking of several receptors for signal generation could solve this problem.