Difference between revisions of "Team:Kent"

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   <h1 align="center"> Welcome to our iGem page </h1>
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   <h1 align="center"> The Problem </h1>
 
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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 <i>b</i><sub>562</sub> that will transport electrons along the amyloid fibrils. The folding of cytochrome <i>b</i><sub>562</sub> 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 <i>b</i><sub>562</sub> nanowire, which together would provide a renewable source of energy that could be used to power small consumer products.</p>
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With rapid technological advancement in the electrical and computing industry there is pressure to increase processing power while downscaling circuitry. The recent discovery of nanowires has fuelled progress in downscaling electrical circuit boards. Currently most circuit boards use copper clad laminates which create large connections and take up a significant amount of space, leading to large boards. Nanowires provide a solution to downscaling. In practical application nano-wires must be in perfect shapes with non-varying cross sections. Despite recent advancements in nanowire production, deformations and imperfections are almost unavoidable.  
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<h1 align="center">How we’re going to do it</h1>
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<h1 align="center">Our Solution</h1>
  
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<p align="center">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 <i>b</i><sub>562</sub> 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 <i>b</i><sub>562</sub> 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 <i>b</i><sub>562</sub> complex must pass the inner membrane to the periplasm via the Sec translocation pathway. Cytochrome <i>b</i><sub>562</sub> requires the cofactor haem to allow correct folding. Cofactors cannot bind before translocation via the Sec pathway, however cytochrome <i>b</i><sub>562</sub> 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.</p>
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<p align="center">Our amyloid nanowire provides an alternative material and method of production, whilst being environmentally friendly and self-assembling. We use an amyloid forming protein, Sup35-NM, which is exported by our engineered E. coli into a solution where the proteins polymerise to form amyloid. Amyloid fibres are well-suited for using as nanowire due to their high heat stability.</p>
 
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<h1 align="center">How we’re going to do it</h1>
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<h1 align="center">Future</h1>
  
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<p align="center">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 <i>b</i><sub>562</sub> 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 <i>b</i><sub>562</sub> 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 <i>b</i><sub>562</sub> complex must pass the inner membrane to the periplasm via the Sec translocation pathway. Cytochrome <i>b</i><sub>562</sub> requires the cofactor haem to allow correct folding. Cofactors cannot bind before translocation via the Sec pathway, however cytochrome <i>b</i><sub>562</sub> 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.</p>
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<p align="center"> In the future it would be possible to engineer the amyloid fibres to bind to the outside of the E.coli cell at specific points. With further engineering it would be possible to insert an electron carrier into the periplasm, allowing electrons from the electron transport chain to be sequestered into the amyloid nanowire directly. This would ultimately produce a self-powering unit that would generate and transport its own electricity that could be used in consumer products, such as mobile phones, eliminating the need for chargers. </p>
 
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Revision as of 10:27, 2 September 2015


iGEM Kent 2015





The Problem

With rapid technological advancement in the electrical and computing industry there is pressure to increase processing power while downscaling circuitry. The recent discovery of nanowires has fuelled progress in downscaling electrical circuit boards. Currently most circuit boards use copper clad laminates which create large connections and take up a significant amount of space, leading to large boards. Nanowires provide a solution to downscaling. In practical application nano-wires must be in perfect shapes with non-varying cross sections. Despite recent advancements in nanowire production, deformations and imperfections are almost unavoidable.































Our Solution

Our amyloid nanowire provides an alternative material and method of production, whilst being environmentally friendly and self-assembling. We use an amyloid forming protein, Sup35-NM, which is exported by our engineered E. coli into a solution where the proteins polymerise to form amyloid. Amyloid fibres are well-suited for using as nanowire due to their high heat stability.




































Future

In the future it would be possible to engineer the amyloid fibres to bind to the outside of the E.coli cell at specific points. With further engineering it would be possible to insert an electron carrier into the periplasm, allowing electrons from the electron transport chain to be sequestered into the amyloid nanowire directly. This would ultimately produce a self-powering unit that would generate and transport its own electricity that could be used in consumer products, such as mobile phones, eliminating the need for chargers.