Team:Kent/Notebook


iGEM Kent 2015

Weekly Meetings

June

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July

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August

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September

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MEETING 1 23/06/15

Attendees

Suparna, Natalie, Moyra, Richard, Robert, James, Alice, Anna, Katarina, Ben, Morena and Mark
  • Overall plan
    • Use the pVS72 plasmid from VS45 strain (ΔCsgAB) and insert it into the pSBIC3 plasmid in MS348 strain (by Gibson assembly or conventional cloning) to be submitted to iGEM. The pVS72 contains an N-terminal CsgA signal sequence attached to Sup35NM (codes the amyloid protein), which is attached to the gene coding for Cytb562. The sup35NM monomers should assemble above the export system on the cell surface. The cytochrome binds haem cofactor, once outside the cell, which will help to conduct electrons. Ultimate goal would be to have the electrons coming from the electron transport chain (ETC) into the biofilm and going to an electron acceptor.
    1. Make lots of competent cells
    2. Miniprep plasmids out of donor strain (VS45). Cut pSBIC3 with EcoRI and pstI, so that it can accept DNA fragment
    3. Cut pVS72 and insert part of it (proteins of interest) into pSB1C3.
  • Modelling
    • Ideas that came up:
    1. Calculate the ideal length of curli, needed for optimum conductance.
    2. Using kinetics, predict how much current can be passed through a biofilm of the nanowire-forming E.coli.
    3. Haem: changing some amino acids in haem and seeing how it affects ratio of oxidation/reduction in fibrils. This could then potentially be used to power a transistor. Modelling amyloid assembly using association and dissociation constants.
    4. Finding the fibril footprint (how many fibrils can be expressed on the cell surface)
    5. Codon optimization ?
  • Validation
    • Possibilities:
    1. Thioflavin T (this dye binds to amyloid proteins)
    2. Measure conductance – consider the logistics involved (how, where, what material to use)
    3. Use of AFF (?) to validate the structure of biobrick.
  • Application of project
    1. Alternative energy
    2. Adding nanowires to existing systems using Geobacter and Shewanella
  • Entrepreneurship competition
    • Possibility of pitching our product. Talk to Gary about patenting once we have a functioning system and idea.

    MEETING 2 30/06/15

    MEETING 3 7/07/15

    MEETING 4 14/07/15

    MEETING 5 21/07/15

    MEETING 6 28/07/15

    MEETING 7 04/08/15

    MEETING 8 11/08/15

    MEETING 9 18/08/15

    MEETING 10 25/08/15

    MEETING 11 01/09/15

    MEETING 12 08/09/15

    MEETING 13 15/09/15

    MEETING 14 22/09/15