IGEM:Imperial/2010/Fast Response module/Activation phosphorylation

Overview of the activation module

Quorum Sensing in B.substilis

The idea is to fuse our split protease on the components of the quorum sensing system of B.substilis. The signal peptide is the product of ComX gene. The sensor is the transmembrane receptor ComP which autophosphorylates(the first half of the split protease will be attached on ComP). ComA is the relay compoment which gets phosphorylated by ComP (the second part of the split protease will be attached on the ComA). When ComP and Coma associate that would produce the active protease.

• Chris D Hirst 12:12, 15 July 2010 (EDT): What causes expression of ComX under normal circumstances in the cell?

Cracking the split Protease (20/07/2010-N.K.)

Based on the review: "Monitoring regulated protein-protein interactions using split TEV", I recognised the following points to be taken into consideration in later experimentation:

In the review the authors created a library of split TEV protease fragments, and screened them for complementation and recapitulation of activity when two binding partners came together. For the rational design of the TEV fragments the following were considered:

• selection of globular domains
• independent folding units were kept intact and the new N-terminus of the C-terminal fragment was located in the unstructured or loop regions
• the catalytic triad of amino acids that constituted the active sitewere never on the same fragment.

So how did they test for complementation? In their experiments the split proteases were fused to a coiled coil followed by a transmembrane domain. The coiled coils had an affinity for each other to hide their hydrophovic patches. When the coils associated the split fragments were brought in close proximity which activated the protease.

The cleavage site for TEV protease is the amino acid sequence ENLYFQ'G

The article also proposes that coupling the split protease activity to another enzyme would result in the amplification of the signal. More precisely, the split protease offers a transient activation. However if this protease activity is used to to activate another enzyme, this would produce a permanent change in our system and also would have a huge amplification effect. An interesting article (Monitoring protein–protein interactions in intact eukaryotic cells by β-galactosidase complementation) talks about split beta-galactosidase enzyme which can be used with x-gal to produce a colour output.

References

• A useful review on quorum sensing. look at the middle of the article for gram positive bacteria Quorum sensing]

• A review of the QS-Competence two componet system of the B.substilis competent and when: regulation of natural genetic competence in bacteria