Difference between revisions of "Team:Kent/Description"

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<h2 align="centre"> Project Description </h2>
 
<h2 align="centre"> Project Description </h2>
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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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Our project aims to produce self-assembling conductive nanowire by harnessing an endogenous amyloid export system in E.coli. This could be used to replace current nanowire technology which relies on chemical synthesis.
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<h2 align="centre">How we're going to do it </h2>
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<li> Our Protein<br>
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<p align="centre">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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The protein that we have chosen to use is Sup35-NM derived from the Sup35 protein found in yeast. Amyloid formation occurs due to stacking interactions of the N domain and each individual Sup35-NM is sufficiently small and closely stacking as to allow the flow of electrons. The amyloid has desirable characteristics for nanostructures, such as heat resistance at both high and low temperatures ranging from 98℃ to -80℃, as well as high mechanical and chemical stability. </li>
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<li> Conduction <br>
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To make our amyloid conductive we have the electron carrier cytochrome b562 bound to Sup35-NM as a fusion protein. Our protein follows the Sec export pathway. This means that our protein will be exported in an unfolded conformation. Type b cytochromes bind haem non-covalently and have four conserved histidine residues that bind to the central iron, thus allowing exogenous addition of haem. To allow electrons to pass to consecutive cytochromes the haem groups must be within 20 Angstroms of each other.  </li>
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<p>Similar work has been carried out harnessing amyloid produced in vitro by a derivative of Sup35-NM with engineered cysteine residues to bind Nanogold particles. This work found that the distance between the Nanogold particles was too large to be conductive and thus a bridging technique was required to reduce of the 3-5nm gap, consequently forcing the diameter of the amyloid to be increased from 9-11nm to 80-200nm (Scheibel, 2003). </p>
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<h2> References </h2>
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<p> Scheibel, T., Parthasarathy, R., Sawicki, G., Lin, X. M., Jaeger, H., & Lindquist, S. L. (2003). Conducting nanowires built by controlled self-assembly of amyloid fibers and selective metal deposition. Proceedings of the National Academy of Sciences, 100(8), 4527-4532.</p>
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Revision as of 10:34, 2 September 2015


iGEM Kent 2015

Project Description

Our project aims to produce self-assembling conductive nanowire by harnessing an endogenous amyloid export system in E.coli. This could be used to replace current nanowire technology which relies on chemical synthesis.

  • Our Protein
    The protein that we have chosen to use is Sup35-NM derived from the Sup35 protein found in yeast. Amyloid formation occurs due to stacking interactions of the N domain and each individual Sup35-NM is sufficiently small and closely stacking as to allow the flow of electrons. The amyloid has desirable characteristics for nanostructures, such as heat resistance at both high and low temperatures ranging from 98℃ to -80℃, as well as high mechanical and chemical stability.
  • Conduction
    To make our amyloid conductive we have the electron carrier cytochrome b562 bound to Sup35-NM as a fusion protein. Our protein follows the Sec export pathway. This means that our protein will be exported in an unfolded conformation. Type b cytochromes bind haem non-covalently and have four conserved histidine residues that bind to the central iron, thus allowing exogenous addition of haem. To allow electrons to pass to consecutive cytochromes the haem groups must be within 20 Angstroms of each other.

    • Similar work has been carried out harnessing amyloid produced in vitro by a derivative of Sup35-NM with engineered cysteine residues to bind Nanogold particles. This work found that the distance between the Nanogold particles was too large to be conductive and thus a bridging technique was required to reduce of the 3-5nm gap, consequently forcing the diameter of the amyloid to be increased from 9-11nm to 80-200nm (Scheibel, 2003).

    References

    Scheibel, T., Parthasarathy, R., Sawicki, G., Lin, X. M., Jaeger, H., & Lindquist, S. L. (2003). Conducting nanowires built by controlled self-assembly of amyloid fibers and selective metal deposition. Proceedings of the National Academy of Sciences, 100(8), 4527-4532.