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>
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<h3>Background</h3>
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<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 E.coli(WT). They play a role of redox control in E.coli.
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<br>&nbsp;&nbsp;&nbsp;&nbsp;Oxidative stress avtivates glutathione reductase (gor) and thioredoxin reductase (trxB). Therefore, E.coli is constantly reductive.<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;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;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;So, we thought changing the E.coli cytosol to oxidative state like yeast leads to be attracted by magnets.<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;That's why knocking out trxB and gor is neccesary to acchive magnetism.
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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>
</p>
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<h3 style="background-color:#ffdead ">2.Materials & Methods</h3>
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<h3 style="background-color:#f0ffff ">2.1.Strain</h3>
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<p>
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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>
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<a href="#">parts</a>
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</p>
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<h3 style="background-color:#f0ffff ">2.2Evaluation of intracellular oxidative state</h3>
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<p>
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<a href="https://2013.igem.org/Team:Chiba/Assay/oxidation">Assay</a>
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</p>
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<h3 style="background-color:#ffdead ">3.Results & Discussion</h3>
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<p>
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</p>
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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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<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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</p>
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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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<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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</p>
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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.
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</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.

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