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>
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  &nbsp;&nbsp;&nbsp;&nbsp;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).<br>
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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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&nbsp;&nbsp;&nbsp;&nbsp;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.
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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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<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;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.)
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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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<br><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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&nbsp;&nbsp;&nbsp;&nbsp;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.<br><br>
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<br>
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&nbsp;&nbsp;&nbsp;&nbsp;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.
 
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<br><br><br>
 
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&nbsp;&nbsp;&nbsp;&nbsp;It is known that Oxidative stress (ex. from ultra violet or oxidant) activates glutathione and thioredoxin.
 
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(Ref. Nakamura H, Nakamura K, Yodoi J: Redox regulation of cellular activation. Ann. Rev. Immuol., 15: 351-369, 1997)<br>
 
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&nbsp;&nbsp;&nbsp;&nbsp;Glutathione and thioredoxin acts as an electron donor, and does reduction of all disulfide bond (formed in cellular protein) into cysteine.<br><BR>
 
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&nbsp;&nbsp;&nbsp;&nbsp; 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.<br><br><br>
 
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  &nbsp;&nbsp;&nbsp;&nbsp;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.<br><br>
 
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&nbsp;&nbsp;&nbsp;&nbsp;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.
 
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<br><br>
 
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&nbsp;&nbsp;&nbsp;&nbsp;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.<br><br>
 
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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>
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<a href="#">Assay</a>
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<a href="https://2013.igem.org/Team:Chiba/Assay/oxidation">Assay</a>
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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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<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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<strong>1.  Strain</strong><br>
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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>
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<center><img src="https://static.igem.org/mediawiki/2013/e/e1/Chiba_MBst.png"></center>
<center><img src="https://static.igem.org/mediawiki/2013/e/e1/Chiba_MBst.png"></center>
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<center><p>Fig n. Evaluation strain of BL21 and Shuffle</p></center><br>
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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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<p>
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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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<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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<h3 style="background-color:#ffdead ">4.Conclusion</h3>
<h3 style="background-color:#ffdead ">4.Conclusion</h3>
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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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We could not distinguish the difference of the redox potential by colorimetric assay using redox indicator, methylene blue.
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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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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"