Team:Chiba/Project/oxidation

From 2013.igem.org

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<h2 id="oxidation" style="background-color:#ff9933"><center>Oxidation</center></h2>
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<h2 id="oxidation" style="background-color:#ff9933"><center>Iron Oxidation</center></h2>
<h3 style="background-color:#ffdead ">1.Introduction</h3>
<h3 style="background-color:#ffdead ">1.Introduction</h3>
<p>
<p>
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&nbsp;&nbsp;&nbsp;&nbsp;Two proteins called glutathione and thioredoxin which have disulfide bond (-s-s-) in oxidized states exist in <i>E.coli</i>(WT). They play a role of redox control in <i>E.coli</i>.
 
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<br>&nbsp;&nbsp;&nbsp;&nbsp;Oxidative stress avtivates glutathione reductase (<i>gor</i>) and thioredoxin reductase (<i>trxB</i>). Therefore, <i>E.coli</i> is constantly reductive.<br>
 
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&nbsp;&nbsp;&nbsp;&nbsp;On the other hand, cytosol of yeast is oxidative originally and they can have magnetism, and be attracted by magnets.<br>
 
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&nbsp;&nbsp;&nbsp;&nbsp;So, we thought changing the <i>E.coli</i> cytosol to oxidative state like yeast leads to be attracted by magnets.<br>
 
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&nbsp;&nbsp;&nbsp;&nbsp;That's why knocking out <i>trxB</i> and <i>gor</i> is neccesary to acchive magnetism.
 
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酵母は，クエン酸鉄を含む培地で培養したとき磁性を持ち、磁石に引き寄せられることがわかっている(Ref.西田論文)。酵母が磁性を持つ主要因はFe3O4の状態の鉄である．他の状態の鉄~例えばXXやYYは，酵母に磁性を与えない。細胞内が酸化的であるときにのみ，鉄はFe3O4の形態をとる。そのため，細胞内の酸化・還元状態は細胞に磁性を与えるための重要なファクターであり，より酸化的な状態が望ましい。酵母にはTCO89という遺伝子が存在し、(TCO89が多いほど細胞は酸化的で少ないほど還元的となるのだが、) この働きによって，酵母細胞内は酸化的な環境が維持されている。一方，大腸菌では，グルタチオンとチオレドキシンという2つのタンパク質が、細胞内の酸化還元状態をコントロールする役割を担っており、この働きによって大腸菌細胞内は還元的な環境が維持されている。ゆえに，大腸菌はほとんど磁性を持たないと考えられる。大腸菌に，磁性を持たせようとするならば，酵母と同じように，酸化的な細胞内環境を作り出す必要があると推測される。グルタチオンとチオレドキシンは、紫外線や酸化剤などによる酸化ストレスによって(発現が)誘導されることが知られている (引用Nakamura H, Nakamura K, Yodoi J: Redox regulationof cellular activation. Ann. Rev. Immuol., 15: 351-369, 1997)。グルタチオンとチオレドキシンは電子供与体として作用することによって、細胞性タンパク質中に形成されるあらゆるジスルフィド結合をシステインに還元する働きをする。一方、NADPHおよび還元酵素であるグルタチオンレダクターゼ(gor)とチオレドキシンジスルフィドレダクターゼ(trxB)は，グルタチオンとチオレドキシンを還元する働きをもつ。この働きによって、基質タンパク質のジスルフィド結合が還元され-SH HSとなることで、細胞内環境が還元的に変化する(Ref.)。このことから，もしgorとtrxBをノックダウンされれば，基質タンパク質のジスルフィド結合が保たれ、そして、大腸菌の細胞内環境が、鉄が磁性をもつに必要な酸化的状態に維持されるのではないかと考えた。
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&nbsp;&nbsp;&nbsp;&nbsp;Nishida <i>et al</i>. first discovered normally diamagnetic yeast Saccharomyces cerevisiae were attracted towards a magnet when grown with ferric citrate<a href="https://2013.igem.org/Team:Chiba/Reference"><sup>1</sup></a>. Because ferromagnetic magnetite (Fe<sub>3</sub>O<sub>4</sub>) 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.<br>
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　大腸菌Shuffle株 (NEB)は、BL21株を，S-S結合を有するタンパク質を正しく発現できるよう改良した株であり、trxB遺伝子が不活性化されている。われわれは、gor, trxB遺伝子の有無によって大腸菌Shuffle株の細胞内が酸化的状態になっていることを、メチレンブルー(酸化還元指示薬)を含む固体培地上に形成させたコロニーの色として評価した。
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&nbsp;&nbsp;&nbsp;&nbsp;On the other hand, in <i>E. coli</i>, there are two proteins called glutathione (<i>gor</i>) and thioredoxin (<i>trxB</i>) play a central role in modulating cellular redox and makes it reductive (detailed mechanisms are described <a href="https://2013.igem.org/Team:Chiba/oxydation/redox homeostasis">here</a>.)<br><br>
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&nbsp;&nbsp;&nbsp;&nbsp;From these facts, we could magnetize <i>E. coli</i> by modulating its redox more oxidative state like in yeast. So, we decided to knock down <i>gor</i> and <i>trx</i> to make cellular redox to more oxidized state, in which iron can form ferromagnetic magnetite (Fe<sub>3</sub>O<sub>4</sub>) and cells are magnetized.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;1. We first used <a href="https://www.neb.com/products/c3025-shuffle-competent-e-coli">SHuffle®</a> (NEB) strain derived from <i>E. coli</i> stain BL21 in which gor and <i>trxB</i> is inactivated. And we compared the redox state of thesb two strains (wild type <i>E. coli</i> (BL21) and <a href="https://www.neb.com/products/c3025-shuffle-competent-e-coli">SHuffle®</a> (NEB)) with a redox indicator, which would change to more blue colour when cellular redox is more oxidized.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;2. Second, we tried to conditionally knock-down these two genes by using CRISPRi system(on going).
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<br>
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</p>
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<h3 style="background-color:#ffdead ">2.Materials&Methods</h3>
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<h3 style="background-color:#ffdead ">2.Materials & Methods</h3>
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<h3 style="background-color:#f0ffff ">2.1plasmid construct</h3>
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<h3 style="background-color:#f0ffff ">2.1.Strain</h3>
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<a href="https://www.neb.com/products/c2530-bl21-competent-e-coli"><i>E. coli</i>(BL21)</a><br>
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<a href="https://www.neb.com/products/c3025-shuffle-competent-e-coli">SHuffle®</a> (NEB)<br>
<a href="#">parts</a>
<a href="#">parts</a>
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<h3 style="background-color:#f0ffff ">2.2細胞内の酸化状態の評価</h3>
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<h3 style="background-color:#f0ffff ">2.2Evaluation of intracellular oxidative state</h3>
<p>
<p>
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<a href="#">Assay</a>
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<a href="https://2013.igem.org/Team:Chiba/Assay/oxidation">Assay</a>
</p>
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<h3 style="background-color:#ffdead ">3.Results&Discussion</h3>
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<h3 style="background-color:#ffdead ">3.Results & Discussion</h3>
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<h3 style="background-color:#f0ffff ">3.1.細胞株</h3>
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<p>
<p>
</p>
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<h3 style="background-color:#f0ffff ">3.2.細胞内の酸化状態の評価</h3>
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<h3 style="background-color:#f0ffff ">3.1.Evaluation of intracellular oxidative state</h3>
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<p>
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<p>
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<strong>Result</strong><br>
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1) We found no significant color difference between the colony of two strains with all concentrations of methylene blue used . <br>
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2) Both strains showed compromised cell growth in the presence of methylene blue presumably because a higher rate of methylene blue reduction interferes with cellular metabolism.<br>
</p>
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<h3 style="background-color:#ffdead ">4.Conclusion</h3>
 
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<center><img src="https://static.igem.org/mediawiki/2013/e/e1/Chiba_MBst.png"></center>
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<center><p><b>Fig. 1</b> Evaluation strain of BL21 and <a href="https://www.neb.com/products/c3025-shuffle-competent-e-coli">SHuffle®</a></p></center><br>
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<table>
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<center><img src="https://static.igem.org/mediawiki/2013/e/e4/Chiba_MB.lift.png"></center>
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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。
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<center><p>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<b>Fig. 2</b> Lift</p></center> 
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　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。
 
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<h3 style="background-color:#ffdead ">4.Conclusion</h3>
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<p>
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We could not distinguish the difference of the redox potential by colorimetric assay using redox indicator, methylene blue.
</p>
</p>

Latest revision as of 03:49, 28 September 2013

iGEM-2013 Chiba

iGEM-2013 Chiba

Iron Oxidation

1.Introduction

    Nishida et al. first discovered normally diamagnetic yeast Saccharomyces cerevisiae were attracted towards a magnet when grown with ferric citrate1. 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 here.)

    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).

2.Materials & Methods

2.1.Strain

E. coli(BL21)
SHuffle® (NEB)
parts

2.2Evaluation of intracellular oxidative state

Assay

3.Results & Discussion

3.1.Evaluation of intracellular oxidative state

Result
1) We found no significant color difference between the colony of two strains with all concentrations of methylene blue used .
2) Both strains showed compromised cell growth in the presence of methylene blue presumably because a higher rate of methylene blue reduction interferes with cellular metabolism.

Fig. 1 Evaluation strain of BL21 and SHuffle®


                Fig. 2 Lift

4.Conclusion

We could not distinguish the difference of the redox potential by colorimetric assay using redox indicator, methylene blue.

Retrieved from "http://2013.igem.org/Team:Chiba/Project/oxidation"