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From 2010.igem.org

Detection Module	Signaling Module	Fast Response Module
We decided to design a new mechanism for parasite detection - by using the proteases they release. A novel protein bound to the cell surface, with a signaling peptide attached via a protease cleavage site. When the protease comes along, the signal peptide is released, allowing it to activate our signaling module.	To transduce the signal we used a quorum sensing system of a gram positive bacterium. The two component signal transduction system taken from S. pneumoniae transfers our peptide signal into the cell, activating the fast response module.	Our fast response mechanism is based around using two enzymatic amplification steps involving a transcripted enzyme, a deactivated enzyme and a presynthesised substrate. This greatly reduces the time required for producing a recognisable output, enabling useful field testing kits.

Using quorum sensing to detect proteases

Having decided that we wanted to detect the parasite protease, we realised that a possible method might be to have it cleave a protein, which would then result in a downstream signalling pathway.

A well-known signalling system using peptides is that of quorum sensing in gram positive bacteria. The signals, or quorum sensing molecules, are known as autoinducing peptides (AIPs) and are transported out of a cell on activation of certain pathways.

AIPs can remain as linear peptides, or they can be post-translationally modified to become circular. We needed to find a system which signals via a linear AIP to eliminate, and one such example is the ComCDE system in Streptococcus pneumoniae which signals via a two component signal transduction system. ComC codes for the competent-stimulating peptide-1 (CSP-1) which is transported out of cells and subsequently detected by a the sensory histidine kinase ComD. ComD then autophosphorylates and then activates the response regulator ComE by phosphorylating it. ComE then binds to two imperfect direct repeats to induce transcription of specific target genes.