Team:Chiba/Project/oxidation

Oxidation

1.Introduction

    Nishida et al. first discovered normally diamagnetic yeast Saccharomyces cerevisiae were attracted towards a magnet when grown with ferric citrate (ref. K. Nishida et al. :Induction of Biogenic Magnetization and Redox Control by a Component of the Target of Rapamycin Complex 1 Signaling Pathway., PLoS Biology, 10, e1001269.). Because ferromagnetic magnetite (Fe₃O₄) is mainly responsible for magnetization, the redox state inside the cell is an important factor. In yeast, mgnetification is further enhanced by TCO89 overexpression , which leads cellular redox to more oxidized state.
    On the other hand, in E. coli, there are two proteins called glutathione (gor) and thioredoxin (trxB) play a central role in modulating cellular redox and makes it reductive (detailed mechanisms are described in next section).
    From these facts, we could magnetize E. coli by modulating its redox more oxidative state like in yeast. So, we decided to knock down gor and trx to make cellular redox to more oxidized state, in which iron can form ferromagnetic magnetite (Fe₃O₄) and cells are magnetized.
    1. We first used SHuffle® (NEB) strain derived from E. coli stain BL21 in which gor and trxB is inactivated. And we compared the redox state of thesb two strains (wild type E. coli (BL21) and SHuffle® (NEB)) with a redox indicator, which would change to more blue colour when cellular redox is more oxidized.
    2. Second, we tried to conditionally knock-down these two genes by using CRISPRi system(on going).

E. coli redox homeostasis
    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.

2.Materials&Methods

2.1plasmid construct

parts

2.2Evaluation of intracellular oxidative state

Assay

3.Results&Discussion

3.1.Strain

3.2.Evaluation of intracellular oxidative state

1. Strain

Fig. n Evaluation strain of BL21 and SHuffle®

2. Lift

Fig. n before lift	Fig. n after lift

Result
1) We found no significant color difference between the colony of two strains with all concentrations of Methylene Blue used .

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。