Team:Chiba/Project/oxidation

Oxidation

1.Introduction

    It is known that when yeast is cultured in a culture that contains ferric citrate, it will be attracted by a magnet (ref. 西田論文).
    The main factor that magnetizes something is iron. Specially, when iron is in form of Fe3O4, it has magnetism. So, the redox state inside the cell is an important matter.

    In yeast, there exists a gene named TCO89. This gene has a function to make cytosol of yeast oxidative, so cytosol of yeast is originally oxidative. (If there is many TCO89 inside the cell, it would be more oxidative inside the cell.)

    On the other hand, in E. coli, there are two proteins called glutathione and thioredoxin. The two proteins play a role of redox control in E. coli, and makes reductive.

    From these facts, we expected that if we make E. coli oxidative like the yeast, it would get magnetized and would be attracted by a magnet.


    It is known that Oxidative stress (ex. from ultra violet or oxidant) activates glutathione and thioredoxin. (Ref. Nakamura H, Nakamura K, Yodoi J: Redox regulation of cellular activation. Ann. Rev. Immuol., 15: 351-369, 1997)
    Glutathione and thioredoxin acts as an electron donor, and does reduction of all disulfide bond (formed in cellular protein) into cysteine.

     Besides, NADPH and the two reductase, glutathione reductase (gor) and thioredoxin-disulfide reductase (trxB) reduces glutathione and thioredoxin. From this, disulfide bond in matrix protein would be reduced into -SH HS and the redox state inside the cell would be reductive.


    Based on these factors, we expected that if we knock down gor and trxB, disulfide bond in matrix protein would be remained and inside E. coli would be oxidant. That means iron can be in form of Fe3O4, and have magnetism.

    Shuffle (NEB) is a transformant of E. coli stain BL21. gor and trxB in this is inactivated, so protein with disulfide bonds could be expressed in it.

    Our team compared the redox state of E. coli that has active gor and trxB with that has inactive gor and trxB, by culturing BL21 (TAKARA) and Shuffle (NEB) with a redox indicator, and observing the change of the color of the colony it forms.

2.Materials&Methods

2.1plasmid construct

parts

2.2細胞内の酸化状態の評価

Assay

3.Results&Discussion

3.1.細胞株

3.2.細胞内の酸化状態の評価

4.Conclusion

操作1.2.3について、shuffleとBL21に劇的な差は見られなかった。考えられることは、shuffle内における酸化的状態がメチレンブルーの変色域に達していないということが考えられる。また、メチレンブルーの毒性によってコロニーのgrowthが悪いところがあった。 　指示薬の変色域とジスルフィド結合の酸化還元ポテンシャルを考慮すること、鉄イオンとジスルフィド結合の酸化還元ポテンシャルを考慮することが今後の課題となる。