Team:Imperial College London/Software Tool

From 2010.igem.org

Software Tool

We realised early on that our detection module could be designed with a sensitivity to different proteases. By changing the cleavage site the system can accept a wide variety of inputs. This tool is designed to facilitate a quick custom sequence generation of the entire surface protein construct.

Select Protease	Description
	This was our primary target. Read our wiki to find out more!

Awaiting sequence generation...
Yellow - Biobrick Prefix/Suffix Orange - Promoter Red - Ribosome Binding Site Violet - Scar Purple - Cell Wall Binding Domain Dark Blue - Adjustable Linker Light Blue - Protease Cleavage Site Dark Green - Autoinducing Peptide Light Green - Terminator

What else could we attach to a cell wall binding domain?

Attaching enzymes to the surface of B. subtilis:

This could be used for a variety of applications including biofuel production. In order for B. subtilis to absorb lignocellulose material, and use it to produce biofuels, this material first needs to be broken down into monosaccharides. Lignocellulose breakdown enzymes, such as cellulase, hemicellulase, and ligninase. could therefore be attached to the surface of B. subtilis.

Flagellin: A Pathogen-associated molecular pattern (PAMP):

PAMPs are sensed by our innate immune system, specifically by pattern-recognition receptors (PRRs). PAMPs are often shared between many different classes of microbes, so they work well as a generic target for the immune system early on in an infection.

Flagellin is one such PAMP and is recognised by a PRR called Toll-like receptor 5 (TLR-5). Flagellin monomers make up flagella of many bacteria, giving them motility which often confers pathogenicity.

Engineering a flagellin monomer to be bound to the exterior of our organism could initiate an immune response, such as the production of the cytokine TNF (tumor necrosis factor) which could help inhibit viral replication in the body.

Antigen presentation:

Antigen presentation is vital when initiating the adaptive immune response. There are many different proteins that we could engineer to be on the surface of our organism, in order for the organism to be phagocytosed by antigen presenting cells (APCs). These APCs would then display the antigen on their surface, held in place by a majorhistocompatability complex (MHC) Class II molecule.

Using this system one could initiate long-term immunity to a variety of pathogens.

DNA binding proteins:

This could be a very useful application for our system because it could be used to bind DNA in the extracellular environment. We could use DNA-binding domains (DBDs) from proteins like transcription factors to bind a specific DNA sequence.

Gene expression libraries:

By attaching each member of a protein library to the surface of a cell, it would be possible to screen for ligands to the proteins. This could be applied to the testing of ligand-binding of proteins after site-directed mutagenesis, allowing the identification of key amino acid residues for ligand binding.