Team:Chiba/Project

From 2013.igem.org

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<h2 id="over" style="background-color:#ff9933"><center>Introduction</center></h2>
<h2 id="over" style="background-color:#ff9933"><center>Introduction</center></h2>
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  <p>&nbsp;&nbsp;&nbsp;&nbsp;Magnetic force is widely used in our life.<br>
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  <p>
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&nbsp;&nbsp;&nbsp;&nbsp;When we listen to the music, we use an earphone or a speaker which uses magnet.<br>
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<strong>Why bother magnetizing?</strong><br>
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&nbsp;&nbsp;&nbsp;&nbsp;When we buy things, we use credit cards that is magnetized.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;We are surrounded by magnets. Magnetized materials are everywhere makes up core components of electrical switches, speaker systems, and portable memory devices (including credit cards), and diagnostic/ separation systems.  Our hope is to program <i>E. coli.</i> cell to turn into magnets to be used for various purposes.<br><br>
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&nbsp;&nbsp;&nbsp;&nbsp;When we watch movies in our house, we use DVD which is magnetized.<br>
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<br>
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Magnet force is unique in that; <br>
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&nbsp;&nbsp;&nbsp;&nbsp;Like this example, magnetic force is used everywhere.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;1. Non-contact (least chance of damaging and contamination)<br>
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<br>
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&nbsp;&nbsp;&nbsp;&nbsp;2. Barrier-free mode of collections (can penetrate the physical blockage)<br>
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&nbsp;&nbsp;&nbsp;&nbsp;With a Magnetic force, we can easily separate things.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;3. Rapidity (immediate exercise)<br>
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&nbsp;&nbsp;&nbsp;&nbsp;In combination with quorum sensing, we can apply Magnetic force to a switch.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;4. Duration (eternal action if needed)<br><br>
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&nbsp;&nbsp;&nbsp;&nbsp;Magnetic force is very useful.<br>
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<br>
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&nbsp;&nbsp;&nbsp;&nbsp;So our team makes <i>E. coli</i> magnetized.<br>
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&nbsp;&nbsp;&nbsp;&nbsp;There are so many good points to use Magnetic force<br>
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</p>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1. You don't have to remove inducer from the reaction system (In the first place you don't need an inducer)<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2. Magnetic force has a transparency (light dosen't)<br>
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3. It reacts immediately<br>
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<br>
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<br></p>
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<div class="section">
<div class="section">
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<h2 id="over" style="background-color:#ff9933"><center>Overview</center></h2>
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<h2 id="over" style="background-color:#ff9933"><center>Overview/Strategies</center></h2>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;Some metal oxides of the spinel type i.e. Fe3O4 have ferrimagnetism because of the disparity of magnetic moment. If <i>E. coli</i> has it, it will be attracted by magnets.
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<p>&nbsp;&nbsp;&nbsp;&nbsp;We have three steps to create magnetic <i>E. coli</i>.<br><br>
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<br>&nbsp;&nbsp;&nbsp;&nbsp;Ordinary, Fe inside cytosol are reduced and exists as divalent ferrous ion. Fe ions, which injure the <i>DNA</i>s with hydroxyl radical from Fenton reaction, are isolated from cytosol by ferritin. Ferritin accumulates ferrous ions inside and oxidizes to ferric ions for prevention of Fenton reaction.
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<strong>1. Reprogramming the iron homeostasis:</strong>To maximize the chance of magnetization, we would like to pump as much Fe into the cell as possible, and keep it. To this end, we tried to eliminate the negative controller (encoded by <i>fur</i>) on the Fec system (iron importer). Also, we tried to knock down the Fe exporter <i>fie</i>F. <br><br>
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<br>&nbsp;&nbsp;&nbsp;&nbsp;<i>Fur</i> controls <i>E. coli</i> iron metabolism. In the case of iron oversupply, fur combine with ferrous ion and restricts the expression of <i>Fec</i>(iron importer) and induces the expression of ferritin. In the other hand, <i>fieF</i> is iron efflux pump which fur doesn't control. These maintain iron homeostasis of <i>E. coli</i>.
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<strong>2. Establishment of Iron storage system:</strong> Fe (II) has severe toxicity to the cell because it casts multiple damages to the DNAs via Fenton reaction. To deal with this problem, we tried below thing; ferritins are cage-shaped multi-subunit proteins for collecting and accumulating ferrous ions (Fe (II)) inside. This way you can well isolate Fe from the cytosolic components.<br><br>
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<br>&nbsp;&nbsp;&nbsp;&nbsp;In our project, (1) we knock out genes, <i>trxB</i> and <i>gor</i> to shift the oxidation state to more oxidized state inside cytosol so that ferrous ions are easier to be oxidized and exist in cytosol. And, (2) human ferritins are overexpressed in order to deal with ferrous ions quickly which haven't be oxidized yet. It will improve iron tolerance of <i>E. coli</i>. (3)We knock down <i>fur</i> and <i>fieF</i> by CRISPRi for the sake of increase the amount of iron uptake.
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<strong>3. Reprogramming the redox state of the cell:</strong> Some metal oxides of the spinel type i.e. Fe<sub>3</sub>O<sub>4</sub> have ferrimagnetism due to the disparity of magnetic moment. To be better attracted by the magnet, higher ratio of Fe (III) over Fe (II) is preferred. To this end, <i>E. coli</i> 's cytosol system will be reprogrammed from reducing to oxidating, by knocking down genes encoding <i>trx</i>B and <i>gor</i> so that ferrous ions are easier to be oxidized and exist in cytosol. <br><br>
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&nbsp;&nbsp;&nbsp;&nbsp;Because most of the genetic operation above mentioned should have significant impact to the cell, we used <a href="https://2013.igem.org/Team:Chiba/Parts#CRISPRi"><strong>CRISPRi technology</strong></a> for the temporal/ transitional knock-out of the target genes above. <br><br>
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<br>
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&nbsp;&nbsp;&nbsp;&nbsp;Also, to speed up the construction of BioBrick, we re-engineered the existing Biobrick (<a href="http://parts.igem.org/Part:BBa_I746908">BBa_I746908</a>) coding Arabinose-triggering GFP generators so that one can quickly replace the GFP with genes of interest using <a href="https://2013.igem.org/Team:Chiba/Parts#golden"><strong>GoldenGate method.</strong></a><br><br>
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<br>We have three steps to create magnetic <i>E. coli</i>.</p>
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<ol style="font-size:18px"><strong>
 
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<li>increase iron uptake</li>
 
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<li>improve iron storage</li>
 
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<li>change the iron redox state</li>
 
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</strong>
 
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</ol>
 
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<p>And, there are each analytical methods.<br><br></p>
 
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<h2 style="background-color:#ff9933"><center>Project</center></h2>
<h2 style="background-color:#ff9933"><center>Project</center></h2>
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<center>
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<table border="1">  
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<td align="center">
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<a href="https://2013.igem.org/Team:Chiba/Project/uptake"><img src = "https://static.igem.org/mediawiki/2013/a/a3/Chiba_storage.jpg" ALT = "#"width="300px"height="400px"></A>
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<a href="https://2013.igem.org/Team:Chiba/Project/uptake"><img src = "https://static.igem.org/mediawiki/2013/archive/c/c2/20130927162646%21Chiba_uptake.jpg" ALT = "#"width="216.5px"height="349px"></A>
</td>
</td>
<td align="center">
<td align="center">
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<a href="https://2013.igem.org/Team:Chiba/Project/store"><img src = "https://static.igem.org/mediawiki/2013/c/c2/Chiba_uptake.jpg" ALT = "#"width="300px"height="400px"></a>
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<a href="https://2013.igem.org/Team:Chiba/Project/store"><img src = "https://static.igem.org/mediawiki/2013/a/a3/Chiba_storage.jpg" ALT = "#"width="211px"height="346.5px"></a>
</td>
</td>
<td align="center">
<td align="center">
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<a href="https://2013.igem.org/Team:Chiba/Project/oxidation"><img src = "https://static.igem.org/mediawiki/2013/8/8b/Chiba_oxidation.jpg" ALT = "#"width="300px"height="450px"></a>
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<a href="https://2013.igem.org/Team:Chiba/Project/oxidation"><img src = "https://static.igem.org/mediawiki/2013/archive/8/8b/20130927163941%21Chiba_oxidation.jpg" ALT = "#"width="218px"height="349.5px"></a>
</td>
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<th>
<th>
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<a href="https://2013.igem.org/Team:Chiba/Project/uptake">Iron Uptake</a>
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<a href="https://2013.igem.org/Team:Chiba/Project/uptake"><strong>Reprogramming the iron <br>homeostasis</strong></a>
</th>
</th>
<th>
<th>
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<a href="https://2013.igem.org/Team:Chiba/Project/store">Iron Storage</a>
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<a href="https://2013.igem.org/Team:Chiba/Project/store"><strong>Establishment of Iron <br>storage system</strong></a>
</th>
</th>
<th>
<th>
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<a href="https://2013.igem.org/Team:Chiba/Project/oxidation">Iron Oxidation</a>
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<a href="https://2013.igem.org/Team:Chiba/Project/oxidation"><strong>Reprogramming the redox state <br>of the cell</strong></a>
</th>
</th>
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</table>
</table>
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</center>

Latest revision as of 04:21, 28 September 2013

iGEM-2013 Chiba

iGEM-2013 Chiba

Introduction

Why bother magnetizing?
    We are surrounded by magnets. Magnetized materials are everywhere makes up core components of electrical switches, speaker systems, and portable memory devices (including credit cards), and diagnostic/ separation systems. Our hope is to program E. coli. cell to turn into magnets to be used for various purposes.

Magnet force is unique in that;
    1. Non-contact (least chance of damaging and contamination)
    2. Barrier-free mode of collections (can penetrate the physical blockage)
    3. Rapidity (immediate exercise)
    4. Duration (eternal action if needed)

Overview/Strategies

    We have three steps to create magnetic E. coli.

1. Reprogramming the iron homeostasis:To maximize the chance of magnetization, we would like to pump as much Fe into the cell as possible, and keep it. To this end, we tried to eliminate the negative controller (encoded by fur) on the Fec system (iron importer). Also, we tried to knock down the Fe exporter fieF.

2. Establishment of Iron storage system: Fe (II) has severe toxicity to the cell because it casts multiple damages to the DNAs via Fenton reaction. To deal with this problem, we tried below thing; ferritins are cage-shaped multi-subunit proteins for collecting and accumulating ferrous ions (Fe (II)) inside. This way you can well isolate Fe from the cytosolic components.

3. Reprogramming the redox state of the cell: Some metal oxides of the spinel type i.e. Fe3O4 have ferrimagnetism due to the disparity of magnetic moment. To be better attracted by the magnet, higher ratio of Fe (III) over Fe (II) is preferred. To this end, E. coli 's cytosol system will be reprogrammed from reducing to oxidating, by knocking down genes encoding trxB and gor so that ferrous ions are easier to be oxidized and exist in cytosol.

    Because most of the genetic operation above mentioned should have significant impact to the cell, we used CRISPRi technology for the temporal/ transitional knock-out of the target genes above.

    Also, to speed up the construction of BioBrick, we re-engineered the existing Biobrick (BBa_I746908) coding Arabinose-triggering GFP generators so that one can quickly replace the GFP with genes of interest using GoldenGate method.

Project

# # #
Reprogramming the iron
homeostasis 		Establishment of Iron
storage system 		Reprogramming the redox state
of the cell

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