Team:Chiba/Project

overview

    Some metal oxides of the spinel type i.e. Fe3O4 have ferrimagnetism because of the disparity of magnetic moment. If E.coli has it, it will be attracted by magnets.
    Ordinary, Fe inside cytosol are reduced and exists as divalent ferrous ion. Fe ions, which injure the DNAs with hydroxyl radical from Fenton reaction, are isolated from cytosol by ferritin. Ferritin accumulates ferrous ions inside and oxidizes to ferric ions for prevention of Fenton reaction.
    Fur controls E.coli iron metabolism. In the case of iron oversupply, fur combine with ferrous ion and restricts the expression of Fec(iron importer) and induces the expression of ferritin. In the other hand, fieF is iron efflux pump which fur doesn’t control. These maintain iron homeostasis of E.coli.
    In our project, (1) we knock out genes, trxB and gor to shift the oxidation state to more oxidized state inside cytosol so that ferrous ions are easier to be oxidized and exist in cytosol. And, (2) human ferritins are overexpressed in order to deal with ferrous ions quickly which haven’t be oxidized yet. It will improve iron tolerance of E.coli. (3)We knock down fur and fieF by CRISPRi for the sake of increase the amount of iron uptake.

We have three steps to create magnetic E.coli.

1. uptake
2. tolerance
3. oxidation

And, there are each analytical methods.

uptake

uptake本文

tolerance

This is .......

oxidation

    E.coli (wild type) can’t form the disulfide bonds because it has reductive state in cytosol.
    We use the shuffle cell whose genes, trxB and gor are knocked out. TrxB and gor codes reductase which … So shuffle cells have oxidative state enough to form the disulfide bonds.