Difference between revisions of "Team:Oxford/Parts"

As our project was to chelate dietary copper we searched for copper binding and storage proteins. We decided that the ideal copper chelator would have these properties: Should be able to bind multiple copper ions per peptide to increase the efficient use of cell resources. They should be from the prokaryotic domain because eukaryotic proteins can have expression issues in E. coli As E. coli naturally deals with copper toxicity by binding copper in the periplasm, periplasmic proteins may reduce toxicity to the host. Using these criteria we found two copper chelators that we though would be useful. We designed gBlocks, codon optimised for E. coli, containing these parts both alone and linked to super-folder GFP. This form of the fluorescent proteins was used because the standard GFP doesn’t fold particularly well in the periplasm where one of our chelators is intended to travel to.

Copper storage protein 1 is a protein discovered in an methane-oxidizing alphaproteobacterium called Methylosinus trichosporium OB3b. (OB3b here stands for “oddball” strain 3b). This bacterium has a high demand for copper for use in its methane monoxygenase enzyme. Vita et al* discovered Csp1 in 2015. Csp1 is a tetramer of four-helix bundles. Each monomer can bind up to 13 Cu(I) ions meaning that the tetramer binds a maximum of 52 copper ions. Vita et al crystallised Csp1 with and without copper bound. The copper is bound inside the pre-folded helical bundles by Cys residues in contrast to metallothioneins which are unstructured until they fold around metal ion clusters. They found an average copper affinity of approximately 1x10^-7M. Csp1 has a signal peptide targeting it to the twin arginine translocation pathway (TAT). This means that it is likely as periplasmic protein. However they also found cytoplasmic homologues in many species challenging their and our assumption that only copper storage occurs in the periplasm due to copper toxicity. We codon optimised Csp1 to E. coli and replaced the original TAT sequence with a TAT sequence from the E. coli protein CueO, which is also involved in copper regulation.

MymT is a small prokaryotic metallothein discovered in Mycobacterium tuberculosis. It can bind up to 7 Cu ions in a solvent shielder core but apparently prefers 4-6. It is much smaller than Csp1 and exists as a monomer. It is cytoplasmic. No crystal structure exists but there is a structure of a eukaryotic homologue:

Each team will make new parts during iGEM and will submit them to the Registry of Standard Biological Parts. The iGEM software provides an easy way to present the parts your team has created. The <groupparts> tag (see below) will generate a table with all of the parts that your team adds to your team sandbox.

Remember that the goal of proper part documentation is to describe and define a part, so that it can be used without needing to refer to the primary literature. Registry users in future years should be able to read your documentation and be able to use the part successfully. Also, you should provide proper references to acknowledge previous authors and to provide for users who wish to know more.

Part Table