Difference between revisions of "Team:Kent"
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Revision as of 14:02, 25 August 2015
Welcome to our iGem page
Our project aims to engineer a novel synthetic biology solution to the production of conductive nano-wires. Our system takes advantage of the endogenous curli system of E.coli to produce functional extracellular amyloid nano-fibrils composed of the amyloid forming domain of the yeast prion protein Sup35NM. The curli system uses the Sec transport pathway to translocate Sup35NM tagged with the Curli-signal sequence through the inner membrane where it is transported from the periplasm through a specific pore to the outer membrane where it assembles into repeating units. We intend to engineer a protein containing Sup35NM (containing the amyloid forming domain of Sup35) linked together to cytochrome b562 that will transport electrons along the amyloid fibrils. The folding of cytochrome b562 requires the cofactor haem, which we are able to add to the growth medium to facilitate folding extra cellular. The wider aim of the project is to funnel electrons from the electron transport chain into the Sup35NM/cytochrome b562 nanowire, which together would provide a renewable source of energy that could be used to power small consumer products.
How we’re going to do it
We are going to utilise the endogenous Curli system of E.coli VS45 strain to export a protein that will form amyloid nano-fibres. The protein will be expressed from a plasmid that we are going to engineer, containing sequence coding for the Sup35NM protein and cytochrome b562 gene inserted into the expression site. The curli system consists of csgG export system, which recognises the csgA N-terminal signal sequence that is attached to the sup35NM protein. This will allow the sup35NM/cytochrome b562 chimera to be translocated to the extracellular space to form an amyloid fibre. To reach the curli-specific pore on the outer membrane the sup35NM/cytochrome b562 complex must pass the inner membrane to the periplasm via the Sec translocation pathway. Cytochrome b562 requires the cofactor haem to allow correct folding. Cofactors cannot bind before translocation via the Sec pathway, however cytochrome b562 can bind to haem that is exogenously added to the solution after export, thus allowing the cytochrome to fold into its active conformation extracellularly. The cytochrome on the amyloid fibres will then transport electrons down the transport chain, acting as a nanowire. We will first create a biofilm of conducting nano-fibres over a surface that we would like electricity to flow across. This will result in a conducting material connecting cells that could be used as a replacement for conventional nanowire allowing the downscaling of consumer products. The ultimate goal of the project is to funnel electrons from the electron transport chain of the bacterial respiratory chain into the amyloid chain. This would allow the production of a self-powering unit that aggregates its own nanowire.