http://2013.igem.org/wiki/index.php?title=Special:Contributions/T.Senda&feed=atom&limit=50&target=T.Senda&year=&month=2013.igem.org - User contributions [en]2024-03-28T18:17:36ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:Chiba/ProjectTeam:Chiba/Project2013-09-28T04:21:54Z<p>T.Senda: </p>
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<h2 id="over" style="background-color:#ff9933"><center>Introduction</center></h2><br />
<p><br />
<strong>Why bother magnetizing?</strong><br><br />
&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><br />
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Magnet force is unique in that; <br><br />
&nbsp;&nbsp;&nbsp;&nbsp;1. Non-contact (least chance of damaging and contamination)<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;2. Barrier-free mode of collections (can penetrate the physical blockage)<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;3. Rapidity (immediate exercise)<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;4. Duration (eternal action if needed)<br><br><br />
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<h2 id="over" style="background-color:#ff9933"><center>Overview/Strategies</center></h2><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;We have three steps to create magnetic <i>E. coli</i>.<br><br><br />
<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><br />
<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><br />
<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><br />
<br />
&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><br />
<br />
&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><br />
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<h2 style="background-color:#ff9933"><center>Project</center></h2><br />
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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><br />
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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><br />
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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><br />
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<a href="https://2013.igem.org/Team:Chiba/Project/uptake"><strong>Reprogramming the iron <br>homeostasis</strong></a><br />
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<th><br />
<a href="https://2013.igem.org/Team:Chiba/Project/store"><strong>Establishment of Iron <br>storage system</strong></a><br />
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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><br />
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</html></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/storeTeam:Chiba/Project/store2013-09-28T04:16:28Z<p>T.Senda: </p>
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<h2 id="storage" style="background-color:#ff9933"><center>Sequestration: Fe-storage machine</center></h2><br />
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<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp; <p><b>Fe must be isolated</b>: In order to magnetize <i>E. coli</i>, we need to stuff as much Fe ions as possible in <i>E. coli</i>. Fe(II) could cause Fenton reaction in response to hydrogen peroxide, harmful hydroxyl radicals (OH•). Dilemma is that, feeding too much Fe into cell would kills the host cell. We decided to over express the ferritins that capture and store Fe irons.</p> <br><br />
<br><br />
<p><b>Fe container machinery</b>: Ferritin particles are made of two small protein subunits (heavy chain (FTH) and light chain (FTL)) (Fig. 1). When expressed, the ferritin subunits automatically assemble into 24-membered protein cages.</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ferrittin.png"alt=""align="middle"></center><br />
<center><p><b>Fig. 1</b> Complex structure of ferritin</p><br></center><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Heavy chain catalyzes oxidation of iron and stimulate 2Fe(II)+O2→[Fe(III)-O-O-Fe(III)] reaction<br />
<br><br />
<br><br />
2Fe<sub>2</sub>+O<sub>2</sub>+(H<sub>2</sub>O)x+3→Fe<sub>2</sub>O<sub>2</sub>(H<sub>2</sub>O)x+4H+H<sub>2</sub>O<sub>2</sub><br />
<br />
<br><br>&nbsp;&nbsp;&nbsp;&nbsp;On the other hand, light chain takes up Fe(III). By functional expression of these two polypeptides, virtual concentration of Fe inside cell can be much reduced. <br />
Interestingly, the storage capacity, the complex size, and FTH/FTL ratio can vary from species to species. Generally speaking, the mammalian ferritin complex contains more FTL than FTH, while the ferritin complex from bacteria have reversed compositions. <br />
<br><br />
<br><br />
<b>Choice of ferritin</b>: Because the storage capacity of <i>E. coli</i> ferritin is far less than that of mammarian type, we decided to make BioBrick for the functional expression of human ferritin in <i>E. coli</i>.<br />
<br><br />
<br><br />
<b>Hypothesis</b>: By storing Fe in isolation, the maximum capacity for Fe storage should be elevated. Also, the apparent iron tolerance of <i>E. coli</i> should be also elevated. <br />
<br />
</br></br><br />
<br><br />
<p><br />
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<h3 style="background-color:#ffdead ">2.Experiments</h3><br />
<br />
<p><br />
<h3 style="background-color:#f0ffff ">2.1.BioBrick construction</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;The ratio of FTH/FTL can be flexible in ferritin complex, and there exist a best composition that gives the highest Fe-storage activity. In heterologous expression of ferritin, the translational efficiency can be fine-tuned so that we could achieve that best composition. So, we constructed BioBricks for the functional expression of human ferritin complex in two formats; <br />
<br><a href="http://parts.igem.org/Part:BBa_K1057002">BBa_K1057002</a>: 'middle' RBS assigned for FTH <br />
<br><a href="http://parts.igem.org/Part:BBa_K1057009">BBa_K1057009</a>: 'strong' RBS assigned for FTH <br />
<br>In both construct, two ferritin genes (FTH and FTL) are placed under pBAD promoter to control the timing and expression level of these genes. To facilitate this construction process, we modified an existing BioBrick (<a href="http://parts.igem.org/Part:BBa_I74608">BBa_I74608</a> deposited by iGEM 2007 team Cambridge) into the new BioBrick(<a href="http://parts.igem.org/Part:BBa_K1057012">BBa_K1057012</a>). This enabled us the rapid, in-parallel, and one-pot digestion/ ligation using Golden gate method. <br />
<br><br />
<br><br />
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<center><img src="https://static.igem.org/mediawiki/2013/2/23/Chiba.ferritin.cloning.png"alt=""align="middle"></center><br />
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<center><p><b>Fig. 2</b> Cloning procedure of ferritin-producing BioBrick</p></center><br><br />
<br><br />
</p><br />
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<a href="https://2013.igem.org/Team:Chiba/Parts"> Parts link</a><br />
</p><br />
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<br />
<h3 style="background-color:#f0ffff ">2.2. Confirmation of Ferritin Expression </h3><br />
<p><br />
Our new BioBricks <a href="http://parts.igem.org/Part:BBa_K1057002">BBa_K1057002</a> and <a href="http://parts.igem.org/Part:BBa_K1057009">BBa_K1057009</a> were transfected into in BL21 strain. The expression level of each individual components (H-chain and L-chain) was checked by SDS-PAGE/ coomassie blue. As a control, we also conducted the same experiment with “sfgfp generator”.<br />
<br><br />
The results are shown in Fig. 3. Both in total protein fraction (gel in left) and soluble fraction(gel in right), there observed two characteristic bands: one was with the size of 20 kDa (corresonding to FTH), while the other was 19kDa (corresponding to FTL). These bands were not detectable for the control sample (Lane 3) expressing the control gene sfGFP under the control of pBAD promoter.<br />
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<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ike.png" width="750px"height="500px"></center><br><br />
<center><p><b>Fig. 3</b> Expression of ferritin in <i>E. coli</i> treated with arabinose. <br />
<br> lane 1. pBAD/araC-ferritin-strong(<a href="http://parts.igem.org/Part:BBa_K1057009">BBa_K1057009</a>)<br />
<br>lane 2. pBAD/araC-ferritin-mid(<a href="http://parts.igem.org/Part:BBa_K1057002">BBa_K1057002</a>)<br />
<br>lane 3. <a href="http://parts.igem.org/Part:BBa_I746908">BBa_I746908</a>(sfgfp)<br />
</p></center><br />
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<h3 style="background-color:#f0ffff ">2.3.Evaluation of iron tolerance</h3><br />
<p><br />
<br />
<strong>Experiment:</strong> <i>E. coli</i> strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with above plasmids. Here, Human ferritin genes (fth1 and ftl) are under the control of pBAD promoter. To express Human ferritin proteins, arabinose (final conc. 0.2%) was added into media. These ferritin generating cell (10^7 cells) inoculated in to fresh media including various concentration of iron citrate (Fe(III)) or iron ascorbate (Fe(II)). After 12h of incubation (at 37ºC), the final cell density and colony forming efficiency was evaluated. As a control, we conducted the same experiment with the cell harboring <a href="http://parts.igem.org/Part:BBa_I746908">BBa_I746908</a> (arabinose-induced sfGFP generator). For the detail of the iron torelance protocol, see <a href="https://2013.igem.org/Team:Chiba/Assay/store">Here. </a><br />
</p><br />
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<h3 style="background-color:#f0ffff ">2.4.Evaluation of magnetism</h3><br />
<p><br />
Now, we finally examined whether BL21 overexpressing ferritin is well attracted to the magnet. Our experimental setup is shown below (Fig. 4). <br> The details of this experimental protocol is given <a href="https://2013.igem.org/Team:Chiba/Assay/store">here</a>.<br />
<center><img src="https://static.igem.org/mediawiki/2013/e/e5/Chiba_magnet_method_ssp.png" width="600px"height="400px"></center><br><br />
<center><p><b>Fig. 4</b> Experimental setup</p></center><br />
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</p><br />
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<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid</h3><br />
<br />
<h3 style="background-color:#f0ffff ">3.2 Assessment of iron tolerance</h3><br />
<br><p><b>Result. 1</b><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/6/6e/Chiba_tetu.jpg" width="744px"height="576px"></center><br><br />
<br><center><p><b>Fig. 5</b> Iron tolerance of <i>E. coli</i> harboring ferritin-expressing BioBricks</p></center><br />
<br><br />
<br></p><p><br />
<br>•Over-expression of ferritin didn’t affect the growth so much. <br><br />
•The cell not expressing overexpression of ferritin, growth inhibition was observed when the concentration of ferrous ascorbate is > 6-8 mM. Virtually, no growth was observed ost cells (both BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a>) above that concentration.<br><br />
<br />
•The cell expressing human ferritins showed significantly higher tolerance to the ferrous ascorbate. <br><br />
<br />
•We observed significant difference in viability profiles of the two strains BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a>. In the case of BL21, expression of ferritins improved the cell growth at semi-inhibitory concentration (5-7 mM) of ferrous ascorbate (up to 10 x), but above that concentration, ferritin expression did not rescue the growth of the cell. In case of <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> strain, the final cell density wasn't that elevated, but it rescued the cell growth at higher concentration (7-8 mM) of ferrous ascorbate. We do not know where this difference comes from. It should be noted that <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> is specifically engineered strain so that the cytosolic environment is more oxidizing than the normal <i>E. coli</i> cells. <br><br />
<br />
•The resultant culture was stamped on the fresh agar plate to form colonies. As is shown below (Fig. 5), The higher tolerance of cells expressing ferritin was again obvious: the cell expressing ferritins retains higher viability (colony-forming capacity) than the control. <br />
<br><center><img src="https://static.igem.org/mediawiki/2013/8/86/Chiba_ike_miracle.png" width="716px"></center><br><br />
<br><center><p><b>Fig. 6</b> Colony-forming capability of <i>E. coli</i> with/without ferritin-expressing BioBricks</p></center><br><br />
<br />
<h3 style="background-color:#f0ffff ">3.3 Evaluation of magnetism</h3><br />
<p><b>Result</b><br><br />
Our expectation was very simple; if E. coli is well magnetized, they should be attracted by magnets so that we should see the emergence of a pattern- due to the accumulation of the cell near the magnet.<br />
Alas!, however, we did not see any trends of E. coli flock forming around magnet: nothing really happened....<br />
<br><br>Maybe, we should have done the same experiment in <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> strain where the cytosolic environment is engineered to be more oxidizing. With the same level of iron contents, the ratio of Fe (III), the required state for ferromagnetism), can be significantly improved. Also, the level of iron accumulation was not sufficient at all. Next step we are trying is to further increase in the cellular iron contents by combining this ferritin over expression system with the temporal knocking down of <i>Fur</i> and <i>fie</i>F using CRISPRi technique. </p><br />
<center><img src="https://static.igem.org/mediawiki/2013/9/93/Chiba_tetsu_BL21_str_ssp.png" width="571px"></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/b/b2/Chiba_tetsu_BL21_mid_sp.png" width="571px"></center><br><br />
<p><br />
<center><p><b>Fig. 7</b> Our first attempt to attract <i>E. coli</i> by magnet.</p></center><br />
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</html></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/storeTeam:Chiba/Project/store2013-09-28T04:13:51Z<p>T.Senda: </p>
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<h2 id="storage" style="background-color:#ff9933"><center>Sequestration: Fe-storage machine</center></h2><br />
<br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp; <p><b>Fe must be isolated</b>: In order to magnetize <i>E. coli</i>, we need to stuff as much Fe ions as possible in <i>E. coli</i>. Fe(II) could cause Fenton reaction in response to hydrogen peroxide, harmful hydroxyl radicals (OH•). Dilemma is that, feeding too much Fe into cell would kills the host cell. We decided to over express the ferritins that capture and store Fe irons.</p> <br><br />
<br><br />
<p><b>Fe container machinery</b>: Ferritin particles are made of two small protein subunits (heavy chain (FTH) and light chain (FTL)) (Fig. 1). When expressed, the ferritin subunits automatically assemble into 24-membered protein cages.</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ferrittin.png"alt=""align="middle"></center><br />
<center><p><b>Fig. 1</b> Complex structure of ferritin</p><br></center><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Heavy chain catalyzes oxidation of iron and stimulate 2Fe(II)+O2→[Fe(III)-O-O-Fe(III)] reaction<br />
<br><br />
<br><br />
2Fe<sub>2</sub>+O<sub>2</sub>+(H<sub>2</sub>O)x+3→Fe<sub>2</sub>O<sub>2</sub>(H<sub>2</sub>O)x+4H+H<sub>2</sub>O<sub>2</sub><br />
<br />
<br><br>&nbsp;&nbsp;&nbsp;&nbsp;On the other hand, light chain takes up Fe(III). By functional expression of these two polypeptides, virtual concentration of Fe inside cell can be much reduced. <br />
Interestingly, the storage capacity, the complex size, and FTH/FTL ratio can vary from species to species. Generally speaking, the mammalian ferritin complex contains more FTL than FTH, while the ferritin complex from bacteria have reversed compositions. <br />
<br><br />
<br><br />
<b>Choice of ferritin</b>: Because the storage capacity of <i>E. coli</i> ferritin is far less than that of mammarian type, we decided to make BioBrick for the functional expression of human ferritin in <i>E. coli</i>.<br />
<br><br />
<br><br />
<b>Hypothesis</b>: By storing Fe in isolation, the maximum capacity for Fe storage should be elevated. Also, the apparent iron tolerance of <i>E. coli</i> should be also elevated. <br />
<br />
</br></br><br />
<br><br />
<p><br />
<br />
<br />
<h3 style="background-color:#ffdead ">2.Experiments</h3><br />
<br />
<p><br />
<h3 style="background-color:#f0ffff ">2.1.BioBrick construction</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;The ratio of FTH/FTL can be flexible in ferritin complex, and there exist a best composition that gives the highest Fe-storage activity. In heterologous expression of ferritin, the translational efficiency can be fine-tuned so that we could achieve that best composition. So, we constructed BioBricks for the functional expression of human ferritin complex in two formats; <br />
<br><a href="http://parts.igem.org/Part:BBa_K1057002">BBa_K1057002</a>: 'middle' RBS assigned for FTH <br />
<br><a href="http://parts.igem.org/Part:BBa_K1057009">BBa_K1057009</a>: 'strong' RBS assigned for FTH <br />
<br>In both construct, two ferritin genes (FTH and FTL) are placed under pBAD promoter to control the timing and expression level of these genes. To facilitate this construction process, we modified an existing BioBrick (<a href="http://parts.igem.org/Part:BBa_I74608">BBa_I74608</a> deposited by iGEM 2007 team Cambridge) into the new BioBrick(<a href="http://parts.igem.org/Part:BBa_K1057012">BBa_K1057012</a>). This enabled us the rapid, in-parallel, and one-pot digestion/ ligation using Golden gate method. <br />
<br><br />
<br><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/2/23/Chiba.ferritin.cloning.png"alt=""align="middle"></center><br />
<br />
<br />
<center><p><b>Fig. 2</b> Cloning procedure of ferritin-producing BioBrick</p></center><br><br />
<br><br />
</p><br />
<br />
<br />
<a href="https://2013.igem.org/Team:Chiba/Parts"> Parts link</a><br />
</p><br />
<br />
<br />
<h3 style="background-color:#f0ffff ">2.2. Confirmation of Ferritin Expression </h3><br />
<p><br />
Our new BioBricks <a href="http://parts.igem.org/Part:BBa_K1057002">BBa_K1057002</a> and <a href="http://parts.igem.org/Part:BBa_K1057009">BBa_K1057009</a> were transfected into in BL21 strain. The expression level of each individual components (H-chain and L-chain) was checked by SDS-PAGE/ coomassie blue. As a control, we also conducted the same experiment with “sfgfp generator”.<br />
<br><br />
The results are shown in Fig. 3. Both in total protein fraction (gel in left) and soluble fraction(gel in right), there observed two characteristic bands: one was with the size of 20 kDa (corresonding to FTH), while the other was 19kDa (corresponding to FTL). These bands were not detectable for the control sample (Lane 3) expressing the control gene sfGFP under the control of pBAD promoter.<br />
<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ike.png" width="750px"height="500px"></center><br><br />
<center><p><b>Fig. 3</b> Expression of ferritin in <i>E. coli</i> treated with arabinose. <br />
<br> lane 1. pBAD/araC-ferritin-strong(<a href="http://parts.igem.org/Part:BBa_K1057009">BBa_K1057009</a>)<br />
<br>lane 2. pBAD/araC-ferritin-mid(<a href="http://parts.igem.org/Part:BBa_K1057002">BBa_K1057002</a>)<br />
<br>lane 3. <a href="http://parts.igem.org/Part:BBa_I746908">BBa_I746908</a>(sfgfp)<br />
</p></center><br />
<br />
<br />
<br />
</p><br />
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<h3 style="background-color:#f0ffff ">2.3.Evaluation of iron tolerance</h3><br />
<p><br />
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<strong>Experiment:</strong> <i>E. coli</i> strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with above plasmids. Here, Human ferritin genes (fth1 and ftl) are under the control of pBAD promoter. To express Human ferritin proteins, arabinose (final conc. 0.2%) was added into media. These ferritin generating cell (10^7 cells) inoculated in to fresh media including various concentration of iron citrate (Fe3+) or iron ascorbate (Fe2+). After 12h of incubation (at 37°C), the final cell density and colony forming efficiency was evaluated. As a control, we conducted the same experiment with the cell harboring <a href="http://parts.igem.org/Part:BBa_I746908">BBa_I746908</a> (arabinose-induced sfGFP generator). For the detail of the iron torelance protocol, see <a href="https://2013.igem.org/Team:Chiba/Assay/store">Here. </a><br />
</p><br />
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<h3 style="background-color:#f0ffff ">2.4.Evaluation of magnetism</h3><br />
<p><br />
Now, we finally examined whether BL21 overexpressing ferritin is well attracted to the magnet. Our experimental setup is shown below (Fig. 4). <br> The details of this experimental protocol is given <a href="https://2013.igem.org/Team:Chiba/Assay/store">here</a>.<br />
<center><img src="https://static.igem.org/mediawiki/2013/e/e5/Chiba_magnet_method_ssp.png" width="600px"height="400px"></center><br><br />
<center><p><b>Fig. 4</b> Experimental setup</p></center><br />
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<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid</h3><br />
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<h3 style="background-color:#f0ffff ">3.2 Assessment of iron tolerance</h3><br />
<br><p><b>Result. 1</b><br />
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<center><img src="https://static.igem.org/mediawiki/2013/6/6e/Chiba_tetu.jpg" width="744px"height="576px"></center><br><br />
<br><center><p><b>Fig. 5</b> Iron tolerance of <i>E. coli</i> harboring ferritin-expressing BioBricks</p></center><br />
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<br>•Over-expression of ferritin didn’t affect the growth so much. <br><br />
•The cell not expressing overexpression of ferritin, growth inhibition was observed when the concentration of ferrous ascorbate is > 6-8 mM. Virtually, no growth was observed ost cells (both BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a>) above that concentration.<br><br />
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•The cell expressing human ferritins showed significantly higher tolerance to the ferrous ascorbate. <br><br />
<br />
•We observed significant difference in viability profiles of the two strains BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a>. In the case of BL21, expression of ferritins improved the cell growth at semi-inhibitory concentration (5-7 mM) of ferrous ascorbate (up to 10 x), but above that concentration, ferritin expression did not rescue the growth of the cell. In case of <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> strain, the final cell density wasn't that elevated, but it rescued the cell growth at higher concentration (7-8 mM) of ferrous ascorbate. We do not know where this difference comes from. It should be noted that <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> is specifically engineered strain so that the cytosolic environment is more oxidizing than the normal <i>E. coli</i> cells. <br><br />
<br />
•The resultant culture was stamped on the fresh agar plate to form colonies. As is shown below (Fig. 5), The higher tolerance of cells expressing ferritin was again obvious: the cell expressing ferritins retains higher viability (colony-forming capacity) than the control. <br />
<br><center><img src="https://static.igem.org/mediawiki/2013/8/86/Chiba_ike_miracle.png" width="716px"></center><br><br />
<br><center><p><b>Fig. 6</b> Colony-forming capability of <i>E. coli</i> with/without ferritin-expressing BioBricks</p></center><br><br />
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<h3 style="background-color:#f0ffff ">3.3 Evaluation of magnetism</h3><br />
<p><b>Result</b><br><br />
Our expectation was very simple; if E. coli is well magnetized, they should be attracted by magnets so that we should see the emergence of a pattern- due to the accumulation of the cell near the magnet.<br />
Alas!, however, we did not see any trends of E. coli flock forming around magnet: nothing really happened....<br />
<br><br>Maybe, we should have done the same experiment in <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> strain where the cytosolic environment is engineered to be more oxidizing. With the same level of iron contents, the ratio of Fe (III), the required state for ferromagnetism), can be significantly improved. Also, the level of iron accumulation was not sufficient at all. Next step we are trying is to further increase in the cellular iron contents by combining this ferritin over expression system with the temporal knocking down of <i>Fur</i> and <i>fie</i>F using CRISPRi technique. </p><br />
<center><img src="https://static.igem.org/mediawiki/2013/9/93/Chiba_tetsu_BL21_str_ssp.png" width="571px"></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/b/b2/Chiba_tetsu_BL21_mid_sp.png" width="571px"></center><br><br />
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<center><p><b>Fig. 7</b> Our first attempt to attract <i>E. coli</i> by magnet.</p></center><br />
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<b>Tasks we couldn't finish until Asia contest</b><br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;•We couldn't double knock down <i>fie</i>F and <i>fur</i> with CRISPRi yet, but we are confident that we can knock down any gene by CRISPRi.<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;•In the not so distant future, we would break <i>E. coli</i> 's iron homeostasis using CRISPRi and would uptake more iron into <i>E. coli</i>.<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;•Even if <i>E. coli</i> 's iron homeostasis would break, over expression of human ferritin could save <i>E. coli</i> alive.<br />
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<br><br><b>Tasks that didn't go like we wanted</b><br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;•Knocking out <i>gor</i> or <i>trx</i>B didn't lead to a grate change of redox potential like TCO89 overexpression in yeast.<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;•It might be hard to oxidize iron in <i>E. coli</i> strain <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a>(It isn't oxidized enough). But by over expression of strong oxidase, maybe we can oxidize iron inside <i>E. coli</i>, make magnetite and magnetize <i>E. coli</i>.<br />
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<br><br><b>What is in future?</b><br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;We decided to design & implement BioBricks that are effective for magnetizing <i>E. coli.</i><br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;If we can freely magnetize any given cells, together with the cell-surface display techniques, you can establish novel systems for bioseparations. Or, you can simplify the harvesting/ collecting step in the process of bioproduction and bioremediation. <br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;<i>E. coli</i> would proliferates infinitely if we feed them and give them iron. So if you use it to a magnetic recording media, perhaps the storage capacity could increase infinitely. <br />
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<h2 id="uptake" style="background-color:#ff9933"><center>Future</center></h2><br><br />
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<p><br />
<b>Tasks we couldn’t finish until Asia contest</b><br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;•We couldn't double knock down <i>fie</i>F and <i>fur</i> with CRISPRi yet, but we are confident that we can knock down any gene by CRISPRi.<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;•In the not so distant future, we would break <i>E. coli</i> 's iron homeostasis using CRISPRi and would uptake more iron into <i>E. coli</i>.<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;•Even if <i>E. coli</i> 's iron homeostasis would break, over expression of human ferritin could save <i>E. coli</i> alive.<br />
<br />
<br><br><b>Tasks that didn't go like we wanted</b><br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;•knocking out <i>gor</i> or <i>trx</i>B didn’t lead to a grate change of redox potential like TCO89 overexpression in yeast.<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;•It might be hard to oxidize iron in <i>E. coli</i> strain <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a>(It isn’t oxidized enough). But by over expression of strong oxidase, maybe we can oxidize iron inside <i>E. coli</i>, make magnetite and magnetize <i>E. coli</i>.<br />
<br />
<br><br><b>What is in future?</b><br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;We decided to design & implement BioBricks that are effective for magnetizing <i>E. coli.</i><br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;If we can freely magnetize any given cells, together with the cell-surface display techniques, you can establish novel systems for bioseparations. Or, you can simplify the harvesting/ collecting step in the process of bioproduction and bioremediation. <br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;<i>E. coli</i> would proliferates infinitely if we feed them and give them iron. So if you use it to a magnetic recording media, perhaps the storage capacity could increase infinitely. <br />
<br />
</p><br />
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</html></div>T.Sendahttp://2013.igem.org/Team:Chiba/NotebookTeam:Chiba/Notebook2013-09-28T04:03:49Z<p>T.Senda: </p>
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<h2 style="background-color:#ff9933"><center>Assay</center></h2><br />
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<a href="https://2013.igem.org/Team:Chiba/Assay/oxidation"><img src = "https://static.igem.org/mediawiki/2013/archive/8/8b/20130927163941%21Chiba_oxidation.jpg" ALT = "#"width="218px"height="349.5px"></a><br />
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<a href="https://2013.igem.org/Team:Chiba/Project/uptake">Reprogramming the iron homeostasis</a><br />
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<a href="https://2013.igem.org/Team:Chiba/Project/store">Establishment of Iron storage system</a><br />
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<h2 id="over" style="background-color:#ff9933"><center>Introduction</center></h2><br />
<p><br />
<strong>Why bother magnetizing?</strong><br><br />
&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><br />
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Magnet force is unique in that; <br><br />
&nbsp;&nbsp;&nbsp;&nbsp;1. Non-contact (least chance of damaging and contamination)<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;2. Barrier-free mode of collections (can penetrate the physical blockage)<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;3. Rapidity (immediate exercise)<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;4. Duration (eternal action if needed)<br><br><br />
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<h2 id="over" style="background-color:#ff9933"><center>Overview/Strategies</center></h2><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;We have three steps to create magnetic <i>E. coli</i>.<br><br><br />
<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><br />
<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><br />
<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><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><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><br />
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<h2 style="background-color:#ff9933"><center>Project</center></h2><br />
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<a href="https://2013.igem.org/Team:Chiba/Project/uptake">Reprogramming the iron homeostasis</a><br />
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<a href="https://2013.igem.org/Team:Chiba/Project/store">Establishment of Iron storage system</a><br />
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<p><center><groupparts>iGEM013 Chiba</groupparts></center></p><br />
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<h2 style="background-color:#ff9933 ">Ferritin</h2><br />
<h3 style="background-color:#ffdead ">Summary</h3><br />
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&nbsp;&nbsp;&nbsp;&nbsp;The ratio of FTH/FTL can be flexible in ferritin complex, and there exist a best composition that gives the highest Fe-storage activity. In heterologous expression of ferritin, the translational efficiency can be fine-tuned so that we could achieve that best composition. So, we constructed BioBricks for the functional expression of human ferritin complex in two formats; <br />
<br><a href="http://parts.igem.org/Part:BBa_K1057002">BBa_K1057002</a>: 'middle' RBS assigned for FTH <br />
<br><a href="http://parts.igem.org/Part:BBa_K1057009">BBa_K1057009</a>: 'strong' RBS assigned for FTH <br />
<br>In both construct, two ferritin genes (FTH and FTL) are placed under pBAD promoter to control the timing and expression level of these genes. To facilitate this construction process, we modified an existing BioBrick (<a href="http://parts.igem.org/Part:BBa_I74608">BBa_I74608</a> deposited by iGEM 2007 team Cambridge) into the new BioBrick(<a href="http://parts.igem.org/Part:BBa_K1057012">BBa_K1057012)</a>. This enabled us the rapid, in-parallel, and one-pot digestion/ ligation using Golden gate method. <br />
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<center><p><b>Fig. 1</B> Cloning procedure of ferritin-producing BioBrick</p></center><br><br />
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<a name="CRISPRi"><h2 style="background-color:#ff9933">CRISPRi</h2></a> <br />
<h3 style="background-color:#ffdead ">Summary</h3><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Recently, Qi and colleagues could show that a nuclease inactive mutant of Cas9 (dCas9) in combination with a sequence specific sgRNA can be utilized for targeted DNA recognition to interfere with transcriptional elongation, RNA polymerase or transcription factor binding (Fig. 2). With unique sgRNA specific for target region, you can knockdown target gene conditionally without any genome modification. This gene silencing activity was termed <strong>CRISPRi</strong> for CRISPR(clustered regularly interspaced short palindromic repeats) interference in reference to RNAi.</p><br />
<br />
<br />
<br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/b/bc/Chiba.CRISPRi.gaiyo.png"alt=""align="middle"></center><br><br />
<p><br />
<center><p><b>Fig. 2</b> CRISPRi mechanism</p></center><br><br />
<br><br />
</P<br />
</p><br />
<br />
<a name="golden" ><h2 style="background-color:#ff9933 "><font size="5.8">Improvement Parts : expression vector with pBAD/Ara switch compatible for<br />
"Golden gate gene swapping"</font></h2></a><br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp; Gentic switch such as pBAD/araC system is very useful for overexpression of given genes. In order to place the various open reading frames with its RBS under the pBAD/araC system, we improved <a href="http://parts.igem.org/Part:BBa_I74608">BBa_I74608</a> to insert BsaI site in both sides of sfgfp gene. This improvement enables us to use ‘Golden Gate’ cloning Method as described below (Fig. 3):<br><br />
1) Preparation of insert fragment : Given gene(s) are PCR amplified with the additional sequence coding for ribosome-binding sites and BsaI site. <br><br />
2) <a href="http://parts.igem.org/Part:BBa_I74608">BBa_I74608</a> and PCR amplified insert fragment is BsaI digested and ligated in a single-pot reaction.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;This method is designed BsaI site doesn't remain on the vector after digesting BsaI. So, you can perform digestion and ligation at the same time. You can obtain desired plasmids in a short time.<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/b/be/Chiba.goldengate.png"alt=""align="middle"></center><br><br />
<br />
<center><p><b>Fig. 3</b> Efficiency of cloning for gene swapping (sfgfp is replaced by mrfp) at different insert/vector molar ration. </p></center><br> <br />
<br />
</p><br />
<h3 style="background-color:#ffdead ">2.Material & Method</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We performed Golden Gate cloning with this part (vector) and mRFP (insert) and checked function. And we investigated the reaction rate changing mol ratio of vector to insert. The protocol is below.<br><br />
1) PCR up insert with BsaI site<br><br />
2) Golden Gate cloning<br><br />
3) transformation<br><br />
Mixture list in Golden Gate is below.<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/7/7c/Chiba.mazehyo.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 4</b> Mixture list </p></center><br> <br />
<br />
<br />
<br />
</p><br />
<h3 style="background-color:#ffdead ">3.Results</h3><br />
<p><br />
<p><br />
1) The highest cloning efficiency (68.9%) was obtained when the insert/vector molar ratio was 1:1. We suppose that the digestion (BsaI) efficiency could decrease with excess amount of insert (resulting in more "non-digested" green fluorescent colonies).<br><br />
2) We found no non-fluorescent colonies in all tested conditions.<br><br />
3) When we want to swap sfgfp to another gene with no phenotypic change, we could screen the right clone as non-fluorescent clones.<br><br />
</p><br />
<br><br />
(A)<br />
<center><img src="https://static.igem.org/mediawiki/2013/4/43/Chiba.graph.png"alt=""align="middle"></center><br />
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<br><br />
(B)<br />
<center><img src="https://static.igem.org/mediawiki/2013/9/92/Chiba.goldengate.plate.png"alt=""align="middle"></center><br />
<br><br />
<center><p><b>Fig. 5</b> Efficiency of cloning for gene swapping(sfgfpto mrfp), (A)percentage of recombinant and non-reconbinant clones (N.D. Not Detected) (B)colony fluorescence<p></center><br />
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</html></div>T.Sendahttp://2013.igem.org/Team:Chiba/Assay/storeTeam:Chiba/Assay/store2013-09-28T03:50:30Z<p>T.Senda: </p>
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<h2>Sequestration Assay</h2><br />
<p><br />
<br />
<br><b>Experiment:</b><br />
<br>Experiment: We constructed two kinds of plasmids with which RBS scores are different as shown in Fig. 2. <i>E. coli</i> strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with above plasmids. Human ferritin genes (fth1 and ftl) are placed on the high-copy plasmid under the control of BAD promoter. To express Human ferritin proteins, arabinose was added into media(final conc. 0.2%). The resultant “ferritin generators” were cultured in the presence of iron citrate (Fe(III)) or iron ascorbate (Fe(II)), and checked final cell density and colony forming efficiency. As a control, we conducted the same experiment with “sfgfp generator”. <br />
<br><br />
<br><b>Detailed procedure</b><br />
<br><br />
1. <i>E. coli</i> strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with the plasmids shown in Fig. 2.<br />
<br><br />
2. All transformants were inoculated into small (2 mL) culture and was shaken for 12h (at 37ºC).<br />
<br><br />
3. Inoculated into flesh media (2 mL), shaken for 3 hours.<br />
<br><br />
4. We added L-arabinose into each sample (final conc. 0.2%). Then cultured for another 9 hours (at 37ºC).<br />
<br><br />
5. After 9 hours, we measured cell density (OD600) of every sample, and put 10<sup>7</sup> cell of <i>E. coli</i> to fresh medium containing various concentration of iron citrate or iron ascorbate.<br />
<br><br />
6. Then we shaking cultured <i>E. coli</i> for 12 hours.<br />
<br><br />
7. We got 1 mL aliquot from each sample and centrifuged with 7,000 rpm, at 25ºC for 2 minutes.<br />
<br><br />
8. We wasted supernatant and resuspended with 1 mL saline solution (0.9% NaClaq), and we measured cell density (OD600).<br />
<br><br />
9. 5 μl of the diluted cell suspension was spotted on the LB agar medium and incubated for 12 hours. Approximately 10<sup>7</sup>, 10<sup>6</sup>, 10<sup>5</sup>, 10<sup>4</sup>, 10<sup>3</sup>, and 10<sup>2</sup> cells were contained in the spots. <br />
<br />
<br />
<br><br><br />
<br />
<br />
</p><br />
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<br />
<br />
<h2>Experiment method of evaluation of magnetism</h2><br />
<p><br><b>Experiment:</b><br><br />
Evaluation of whether <i>E. coli</i> have magnetism by ferritin with iron.<br><br />
<br><br />
<b>Detailed procedure</b><br><br />
Plasmid:pBAD/araC-ferritin-strong(<a href="http://parts.igem.org/Part:BBa_K1057009">BBa_K1057009</a>), pBAD/araC-ferritin-mid(<a href="http://parts.igem.org/Part:BBa_K1057002">BBA_K1057002</a>)<br />
<br><br />
<br>1. Introduction of pBAD/araC-ferritin-strong(<a href="http://parts.igem.org/Part:BBa_K1057009">BBa_K1057009</a>) or pBAD/araC-ferritin-mid(<a href="http://parts.igem.org/Part:BBa_K1057002">BBA_K1057002</a>) into BL21 strain. Preparation of medium(arabinose:final conc. 0.2%, ferric citrate:final conc.10mM). Applying cultured medium to be OD 10and final volume 2 mL.<br />
<br>2.Culturing at 37ºC for an hour.<br />
<br>3.an hour later, the solution diluted(10 fold) by LB agar medium to be final volume 10mL.<br />
<br>4, The plate in 4ml 0.01%triton X-100 solution all over the bottom was added 10ml solution over it.<br><br />
<br>5, 300 mT Doughnut type magnets ware put under the plate, we take pictures at intervals of 5 minutes till 25 minutes later.<br><br />
<br />
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</html></div>T.Sendahttp://2013.igem.org/Team:Chiba/NotebookTeam:Chiba/Notebook2013-09-28T03:36:16Z<p>T.Senda: </p>
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<h2 style="background-color:#ff9933"><center>Assay</center></h2><br />
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<a href="https://2013.igem.org/Team:Chiba/Assay/uptake"><img src = "https://static.igem.org/mediawiki/2013/archive/c/c2/20130927162646%21Chiba_uptake.jpg" ALT = "#"width="216.5px"height="349px"></A><br />
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<a href="https://2013.igem.org/Team:Chiba/Assay/store"><img src = "https://static.igem.org/mediawiki/2013/a/a3/Chiba_storage.jpg" ALT = "#"width="211px"height="346.5px"></a><br />
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<a href="https://2013.igem.org/Team:Chiba/Assay/oxidation"><img src = "https://static.igem.org/mediawiki/2013/archive/8/8b/20130927163941%21Chiba_oxidation.jpg" ALT = "#"width="218px"height="349.5px"></a><br />
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<a href="https://2013.igem.org/Team:Chiba/Project/uptake">Iron Uptake</a><br />
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<a href="https://2013.igem.org/Team:Chiba/Project/store">Iron Storage</a><br />
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<a href="https://2013.igem.org/Team:Chiba/Project/oxidation">Iron Oxidation</a><br />
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</html></div>T.Sendahttp://2013.igem.org/Team:Chiba/Assay/storeTeam:Chiba/Assay/store2013-09-28T03:33:20Z<p>T.Senda: </p>
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<h2>Storage Assay</h2><br />
<p><br />
<br />
<br><b>Experiment:</b><br />
<br>Experiment: We constructed two kinds of plasmids with which RBS scores are different as shown in Fig. 2. E. coli strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with above plasmids. Human ferritin genes (fth1 and ftl) are placed on the high-copy plasmid under the control of BAD promoter. To express Human ferritin proteins, arabinose was added into media(final conc. 0.2%). The resultant “ferritin generators” were cultured in the presence of iron citrate (Fe3+) or iron ascorbate (Fe2+), and checked final cell density and colony forming efficiency. As a control, we conducted the same experiment with “sfgfp generator”. <br />
<br><br />
<br><b>Detailed procedure</b><br />
<br><br />
1. E. coli strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with the plasmids shown in Fig. 1.<br />
<br><br />
2. All transformants were inoculated into small (2 mL) culture and was shaken for 12h (at 37ºC).<br />
<br><br />
3. Inoculated into flesh media (2 mL), shaken for 3 hours.<br />
<br><br />
4. We added L-arabinose into each sample (final conc. 0.2%). Then cultured for another 9 hours (at 37ºC).<br />
<br><br />
5. After 9 hours, we measured cell density (OD600) of every sample, and put 10<sup>7</sup> cell of <i>E. coli</i> to fresh medium containing various concentration of iron citrate or iron ascorbate.<br />
<br><br />
6. Then we shaking cultured <i>E. coli</i> for 12 hours.<br />
<br><br />
7. We got 1 mL aliquot from each sample and centrifuged with 7,000 rpm, at 25ºC for 2 minutes.<br />
<br><br />
8. We wasted supernatant and resuspended with 1 mL saline solution (0.9% NaClaq), and we measured cell density (OD600).<br />
<br><br />
9. 5 μl of the diluted cell suspension was spotted on the LB agar medium and incubated for 12 hours. Approximately 10<sup>7</sup>, 10<sup>6</sup>, 10<sup>5</sup>, 10<sup>4</sup>, 10<sup>3</sup>, and 10<sup>2</sup> cells were contained in the spots. <br />
<br />
<br />
<br><br><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<h2>Experiment method of evaluation of magnetism</h2><br />
<p><br><b>Experiment:</b><br><br />
Ferritinにより鉄を取り込んだ大腸菌が磁性を持つかを確認する<br><br />
<br><br />
<b>Detailed procedure</b><br><br />
Plasmid:pBAD/araC-ferritin-strong(<a href="http://parts.igem.org/Part:BBa_K1057009">BBa_K1057009</a>), pBAD/araC-ferritin-mid(<a href="http://parts.igem.org/Part:BBa_K1057002">BBA_K1057002</a>)<br />
<br><br />
<br><br />
<br><br />
1. Introduction of pBAD/araC-ferritin-strong(<a href="http://parts.igem.org/Part:BBa_K1057009">BBa_K1057009</a>) or pBAD/araC-ferritin-mid(<a href="http://parts.igem.org/Part:BBa_K1057002">BBA_K1057002</a>) into BL21 strain.<br />
2. preparation of medium(arabinose:final concentration <br />
3.<br />
4.<br />
5.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
1, 上に示す2つのプラスミドを導入した大腸菌を、それぞれ試験管に、OD10、終濃度0.2% arabinose、かつ終濃度10mM ferric citrateになるよう、2mlの溶液を調整した。<br><br />
2, 37℃で一時間、培養した。<br><br />
3, 一時間後、溶液を、LB培地により10倍に希釈し、20mlの溶液を調整した。<br><br />
4, The plate in 4ml 0.01%triton X-100 solution all over the bottom was added 10ml solution over it.<br><br />
5, 300 mT Doughnut type magnets ware put under the plate, we take pictures at intervals of 5 minutes till 25 minutes later.<br><br />
<br />
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</body><br />
</html></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/uptakeTeam:Chiba/Project/uptake2013-09-28T03:03:47Z<p>T.Senda: </p>
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<h2 id="uptake" style="background-color:#ff9933"><center>Reprogramming Iron Homeostasis</center></h2><br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;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 <i>Fec</i> system (iron importer). Also, we tried to knock down the Fe exporter <i>fie</i>F. </p><br />
<br />
<h3 style="background-color:#f0ffff ">Target-1:<i>fur</i></h3><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;<i>Fur</i> (Ferric uptake regulator) controls multiple stages of iron metabolism such as taking iron in or storing iron. Major mode-of-action is in binding to <i>Fur</i> box to regulate transcription of various genes involved in the iron homeostasis.<br />
When cellular level of iron increase, <i>Fur</i> turns into the activated form, thereby start restricting the reproduction of the iron transporter (Fec system). Long story short, it ends up with down-regulating the iron uptake. At the same time, this active form <i>Fur</i> negatively controls the expression ofRyhB. RyhB is one of the sRNA that represses the expression of endogenous Ferritin. So, turning <i>Fur</i> in active form (by increasing cellular iron concentration) leads to the induction of ferritin construction.<br><br><br><br />
<p><br />
When iron level get back, <i>Fur</i> turn back to inactive state, thereby restoring the iron uptaking machineries. RyhB would also work normally, so expression of ferritin is down-regulated to prevent iron depletion. On the other hand, other iron importing system EfeUOB recognizes rather ascorbic type and more specialized in importing Fe(II). EfeUOB is also controlled by <i>Fur</i>: When the level of Fe(II) becomes too high, <i>Fur</i> restricts EfeUOB and block uptaking the iron (II).<br />
<br><br><br><br />
</p><br />
<br />
<p><br />
In summary, fur plays important roles iron homeostasis, and it is by nature a negative regulator for iron update. We would like to knockdown Fur so that iron transporter system would stay active even at the 'too much' situation. Possible downside could be that it also reduce the endogenous Ferritin formation, but we are constructing artificial (arabinose-inducible) system to functionally express human ferritins.<br><br><br><br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<h3 style="background-color:#f0ffff ">1.2.<i>fie</i>F</h3><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;There is a Regulator called <i>fie</i>F (ferric iron efflux). It makes iron and zinc flow out of cell and controls detoxification of cell. When <i>fie</i>F is knocked down/out, the tolerance of cell to iron would be lowered.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;<i>Fec</i> has a character that can recognize ferric citrate and taking Fe(III) in. <i>Fec</i> is controlled by <i>Fur</i> and when the density of Fe(III) in the cell is too high, <i>Fur</i> restricts Fec and stops iron uptake.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In the same way, <i>Efe</i>UOB has a character that can recognize ascorbic acid and taking Fe(II) in. <i>Efe</i>UOB is also controlled by <i>Fur</i> and when the density of Fe(II) in the cell is too high, <i>Fur</i> restricts <i>EfeUOB</i> and stops iron uptake.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Using these characters, and by knocking down/out <i>Fur</i> and <i>fie</i>F, <i>Fec</i>/<i>Efe</i>UOB expression wouldn't be restricted so the amount of iron coming in would increase and the amount of iron going out would decrease. The system would work like when iron is lacking.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;As a result, the amount of iron inside <i>E. coli</i> would increase.</p><br />
<br><center><img src="https://static.igem.org/mediawiki/2013/f/f8/Uptake_image.PNG" width="723px"height="305px"></center><br><br />
<br />
</p><br />
<br />
<br />
<h3 style="background-color:#ffdead ">2.Materials & Methods</h3><br />
<br />
<h3 style="background-color:#f0ffff ">2.1.plasmid construct</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We constructed four plasmids knocking down <i>fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. at the same time, in order to confirm the function of dCas9, we constructed a plasmid knocking down <i>lacZ</i>.<br><br />
<br><br />
<br />
<br />
<br />
<a href="#">Part link</a><br><br><br />
</p><br />
<h3 style="background-color:#f0ffff ">2.2.Evaluation of <i>Fur</i>,<i>fie</i>F knockdown</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We performed two experiments about CRISPRi system in order to confirm the knockdown function as desired. The purpose is a function check for <i>lacZ</i>, <i>Fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. <br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/7/7f/Chiba.CRISPRi.kan.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 1</b> the method of function check about <i>fur</i></p></center><br />
<br />
<br />
<br />
<a href="https://2013.igem.org/Team:Chiba/Assay/uptake">Assay</a><br />
</p><br />
<h3 style="background-color:#f0ffff ">2.3.Evaluation of absorbed iron volume</h3><br />
<p><br />
<b>Experiment:</b><br><br />
<br />
<br />
<left><img src="https://static.igem.org/mediawiki/2013/3/3f/CRISPRi.PNG" width="409px"height="88px"></left><br />
<right><img src="https://static.igem.org/mediawiki/2013/2/23/DCas9.PNG" width="306px"height="110px"></right><br><br />
<center><b>Fig. a</b></center><br />
<br><p>&nbsp;&nbsp;&nbsp;&nbsp;<i>E. coli</i> stain BL21 was transformed by Plasmid shown in <b>Fig. a</b> .&nbsp;Then we cultured all transformants with atC.&nbsp; atC was added to knock down <i>fur</i> and <i>fie</i>F.&nbsp; After that we cultured it in the presence of ferric citrate, and measured the density of iron that weren’t taken in to <i>E. coli</i> and remained in the medium by measuring Absorbance (Abs 756) with color reagent.</p><br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<p><br />
<br />
<br />
<h3 style="background-color:#f0ffff ">3.1.Function Check</h3><br />
<p><br />
1) In the absence of anhydro Tetracycline (aTc), CRISPRi-medeated <i>lac</i>Z gene transcriptional silencing did not occur, resulting in blue-colored colony in which <i>lac</i>Z was expressed.<br><br />
2) In the presence of aTc, CRISPRi-medeated <i>lac</i>Z gene transcriptional silencing successfully occurred, resulting in colorless colony in which <i>lac</i>Z was not expressed.<br><br />
3) CRISPRi-medeated <i>fur</i> gene transcriptional silencing successfully occurred, resulting in the loss of kanamysin resistance.<br><br />
</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/26/Chiba.CRISPRi.spot.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 2 </b>CRISPRi efficiently silence transcription</p></center><br><br />
<br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/a/a6/Chiba.CRISPRi-lacZ.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 3</b> Function check about CRISPRi-<i>lacZ</i></p></center><br><br />
<br />
<br />
<br><br />
<br />
<br />
<br />
<br />
<h3 style="background-color:#f0ffff ">3.2.A knockdown of <i>fie</i>F or <i>fur</i> has no effect on iron uptake </h3><br />
<br />
<p><br />
Iron uptake was not detectable (iron concentration changes in media were less than micro molar order), even if <i>fur</i> or <i>fie</i>F was successfully knocked down.<br><br />
A future subject is to experiment at more cell number of <i>E. coli</i>.<br><br />
</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/0/0d/Chiba_shimamura_super.png"width="800px"></center><br><br />
<center><p><b>Fig. 4 </b>Absorbance at each cell number of <i>E. coli</i>(BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> introduced each plusmid</p></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/a/a2/Chiba_shimamura_ultra.png"width="800px"></center><br><br />
<center><p><b>Fig. 5</b> Absorbance of as a function of each iron concentration</p></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/3/39/Chiba_torikomi.png"width="387px"height="537px"></center><br><br />
<center><p><b>Fig. 6</b></p></center><br><br />
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</html></div>T.Sendahttp://2013.igem.org/File:Chiba_shimamura_ultra.pngFile:Chiba shimamura ultra.png2013-09-28T02:57:45Z<p>T.Senda: </p>
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<div></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/storeTeam:Chiba/Project/store2013-09-28T02:51:58Z<p>T.Senda: </p>
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<h2 id="storage" style="background-color:#ff9933"><center>Sequestration: Fe-storage machine</center></h2><br />
<br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp; <p><b>Fe must be isolated</b>: In order to magnetize <i>E. coli</i>, we need to stuff as much Fe ions as possible in <i>E. coli</i>. Fe(II) could cause Fenton reaction in response to hydrogen peroxide, harmful hydroxyl radicals (OH•). Dilemma is that, feeding too much Fe into cell would kills the host cell. We decided to over express the ferritins that capture and store Fe irons.</p> <br><br />
<br><br />
<p><b>Fe container machinery</b>: Ferritin particles are made of two small protein subunits (heavy chain (FTH) and light chain (FTL)) (Fig. 1). When expressed, the ferritin subunits automatically assemble into 24-membered protein cages.</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ferrittin.png"alt=""align="middle"></center><br />
<center><p><b>Fig. 1</b> Complex structure of ferritin</p><br></center><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Heavy chain catalyzes oxidation of iron and stimulate 2Fe(II)+O2→[Fe(III)-O-O-Fe(III)] reaction<br />
<br><br />
<br><br />
2Fe<sub>2</sub>+O<sub>2</sub>+(H<sub>2</sub>O)x+3→Fe<sub>2</sub>O<sub>2</sub>(H<sub>2</sub>O)x+4H+H<sub>2</sub>O<sub>2</sub><br />
<br />
<br><br>&nbsp;&nbsp;&nbsp;&nbsp;On the other hand, light chain takes up Fe(III). By functional expression of these two polypeptides, virtual concentration of Fe inside cell can be much reduced. <br />
Interestingly, the storage capacity, the complex size, and FTH/FTL ratio can vary from species to species. Generally speaking, the mammalian ferritin complex contains more FTL than FTH, while the ferritin complex from bacteria have reversed compositions. <br />
<br><br />
<br><br />
<b>Choice of ferritin</b>: Because the storage capacity of <i>E. coli</i> ferritin is far less than that of mammarian type, we decided to make BioBrick for the functional expression of human ferritin in <i>E. coli</i>.<br />
<br><br />
<br><br />
<b>Hypothesis</b>: By storing Fe in isolation, the maximum capacity for Fe storage should be elevated. Also, the apparent iron tolerance of <i>E. coli</i> should be also elevated. <br />
<br />
</br></br><br />
<br><br />
<p><br />
<br />
<br />
<h3 style="background-color:#ffdead ">2.Experiments</h3><br />
<br />
<p><br />
<h3 style="background-color:#f0ffff ">2.1.BioBrick construction</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;The ratio of FTH/FTL can be flexible in ferritin complex, and there exist a best composition that gives the highest Fe-storage activity. In heterologous expression of ferritin, the translational efficiency can be fine-tuned so that we could achieve that best composition. So, we constructed BioBricks for the functional expression of human ferritin complex in two formats; <br />
<br>BBa_K1057002: 'middle' RBS assigned for FTH <br />
<br>BBa_K1057009: 'strong' RBS assigned for FTH <br />
<br>In both construct, two ferritin genes (FTH and FTL) are placed under pBAD promoter to control the timing and expression level of these genes. To facilitate this construction process, we modified an existing Biobrick (BBa_I74608 deposited by iGEM 2007 team Cambridge) into the new BioBrick(BBa_K1057012). This enabled us the rapid, in-parallel, and one-pot digestion/ ligation using Golden gate method. <br />
<br><br />
<br><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/3/32/Chiba.ferritin.png"alt=""align="middle"></center><br />
<br />
<br />
<center><p><b>Fig. 2</b> Cloning procedure of ferritin-producing BioBrick</p></center><br><br />
<br><br />
</p><br />
<br />
<br />
<a href="https://2013.igem.org/Team:Chiba/Parts"> Parts link</a><br />
</p><br />
<br />
<br />
<h3 style="background-color:#f0ffff ">2.2. Confirmation of Ferritin Expression </h3><br />
<p><br />
Our new BioBricks BBa_K1057002 and BBa_K1057009 were transfected into in BL21 strain. The expression level of each individual components (H-chain and L-chain) was checked by SDS-PAGE/ coomassie blue. As a control, we also conducted the same experiment with “sfgfp generator”.<br />
<br><br />
The results are shown in Fig. 3. Both in total protein fraction (gel in left) and soluble fraction(gel in right), there observed two characteristic bands: one was with the size of 20 kDa (corresonding to FTH), while the other was 19kDa (corresponding to FTL). These bands were not detectable for the control sample (Lane 3) expressing the control gene sfGFP under the control of pBAD promoter.<br />
<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ike.png" width="750px"height="500px"></center><br><br />
<center><p><b>Fig. 3</b> Expression of ferritin in <i>E. coli</i> treated with arabinose. <br />
<br> lane 1. pBAD/araC-ferritin-strong(BBa_K1057009)<br />
<br>lane 2. pBAD/araC-ferritin-mid(BBA_K1057002)<br />
<br>lane 3. BBa_I746908(sfgfp)<br />
</p></center><br />
<br />
<br />
<br />
</p><br />
<br />
<h3 style="background-color:#f0ffff ">2.3.Evaluation of iron tolerance</h3><br />
<p><br />
<br />
<strong>Experiment:</strong> <i>E. coli</i> strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with above plasmids. Here, Human ferritin genes (fth1 and ftl) are under the control of BAD promoter. To express Human ferritin proteins, arabinose (final conc. 0.2%) was added into media. These ferritin generating cell (10^7 cells) inoculated in to fresh media including various concentration of iron citrate (Fe3+) or iron ascorbate (Fe2+). After 12h of incubation (at 37°C), the final cell density and colony forming efficiency was evaluated. As a control, we conducted the same experiment with the cell harboring BBa_I746908 (arabinose-induced sfGFP generator). For the detail of the iron torelance protocol, see <a href="https://2013.igem.org/Team:Chiba/Assay/store">Here. </a><br />
</p><br />
<br />
<h3 style="background-color:#f0ffff ">2.4.Evaluation of magnetism</h3><br />
<p><br />
Now, we finally examined whether BL21 overexpressing ferritin is well attracted to the magnet. Our experimental setup is shown below (Fig. 4). <br> The details of this experimental protocol is given <a href="https://2013.igem.org/Team:Chiba/Assay/store">here</a>.<br />
<center><img src="https://static.igem.org/mediawiki/2013/e/e5/Chiba_magnet_method_ssp.png" width="600px"height="400px"></center><br><br />
<center><p><b>Fig. 4</b> Experimental setup</p></center><br />
<br />
<br><br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid</h3><br />
<br />
<h3 style="background-color:#f0ffff ">3.2 Assessment of iron tolerance</h3><br />
<br><p><b>Result. 1</b><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/6/6e/Chiba_tetu.jpg" width="744px"height="576px"></center><br><br />
<br><center><p><b>Fig. 5</b> Iron tolerance of <i>E. coli</i> harboring ferritin-expressing BioBricks</p></center><br />
<br><br />
<br></p><p><br />
<br>•Over-expression of ferritin didn’t affect the growth so much. <br><br />
•The cell not expressing overexpression of ferritin, growth inhibition was observed when the concentration of ferrous ascorbate is > 6-8 mM. Virtually, no growth was observed ost cells (both BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a>) above that concentration.<br><br />
<br />
•The cell expressing human ferritins showed significantly higher tolerance to the ferrous ascorbate. <br><br />
<br />
•We observed significant difference in viability profiles of the two strains BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a>. In the case of BL21, expression of ferritins improved the cell growth at semi-inhibitory concentration (5-7 mM) of ferrous ascorbate (up to 10 x), but above that concentration, ferritin expression did not rescue the growth of the cell. In case of <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> strain, the final cell density wasn't that elevated, but it rescued the cell growth at higher concentration (7-8 mM) of ferrous ascorbate. We do not know where this difference comes from. It should be noted that <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> is specifically engineered strain so that the cytosolic environment is more oxidizing than the normal <i>E. coli</i> cells. <br><br />
<br />
•The resultant culture was stamped on the fresh agar plate to form colonies. As is shown below (Fig. 5), The higher tolerance of cells expressing ferritin was again obvious: the cell expressing ferritins retains higher viability (colony-forming capacity) than the control. <br />
<br><center><img src="https://static.igem.org/mediawiki/2013/8/86/Chiba_ike_miracle.png" width="716px"></center><br><br />
<br><center><p><b>Fig. 6</b> Colony-forming capability of <i>E. coli</i> with/without ferritin-expressing BioBricks</p></center><br><br />
<br />
<h3 style="background-color:#f0ffff ">3.3 Evaluation of magnetism</h3><br />
<p><b>Result</b><br><br />
Our expectation was very simple; if E. coli is well magnetized, they should be attracted by magnets so that we should see the emergence of a pattern- due to the accumulation of the cell near the magnet.<br />
Alas!, however, we did not see any trends of E. coli flock forming around magnet: nothing really happened....<br />
<br><br>Maybe, we should have done the same experiment in <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> strain where the cytosolic environment is engineered to be more oxidizing. With the same level of iron contents, the ratio of Fe (III), the required state for ferromagnetism), can be significantly improved. Also, the level of iron accumulation was not sufficient at all. Next step we are trying is to further increase in the cellular iron contents by combining this ferritin over expression system with the temporal knocking down of <i>Fur</i> and <i>fie</i>F using CRISPRi technique. </p><br />
<center><img src="https://static.igem.org/mediawiki/2013/9/93/Chiba_tetsu_BL21_str_ssp.png" width="571px"></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/b/b2/Chiba_tetsu_BL21_mid_sp.png" width="571px"></center><br><br />
<p><br />
<center><p><b>Fig. 7</b> Our first attempt to attract <i>E. coli</i> by magnet.</p></center><br />
</p><br />
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</html></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/storeTeam:Chiba/Project/store2013-09-28T02:49:47Z<p>T.Senda: </p>
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<h2 id="storage" style="background-color:#ff9933"><center>Sequestration: Fe-storage machine</center></h2><br />
<br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp; <p><b>Fe must be isolated</b>: In order to magnetize <i>E. coli</i>, we need to stuff as much Fe ions as possible in <i>E. coli</i>. Fe(II) could cause Fenton reaction in response to hydrogen peroxide, harmful hydroxyl radicals (OH•). Dilemma is that, feeding too much Fe into cell would kills the host cell. We decided to over express the ferritins that capture and store Fe irons.</p> <br><br />
<br><br />
<p><b>Fe container machinery</b>: Ferritin particles are made of two small protein subunits (heavy chain (FTH) and light chain (FTL)) (Fig. 1). When expressed, the ferritin subunits automatically assemble into 24-membered protein cages.</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ferrittin.png"alt=""align="middle"></center><br />
<center><p><b>Fig. 1</b> Complex structure of ferritin</p><br></center><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Heavy chain catalyzes oxidation of iron and stimulate 2Fe(II)+O2→[Fe(III)-O-O-Fe(III)] reaction<br />
<br><br />
<br><br />
2Fe<sub>2</sub>+O<sub>2</sub>+(H<sub>2</sub>O)x+3→Fe<sub>2</sub>O<sub>2</sub>(H<sub>2</sub>O)x+4H+H<sub>2</sub>O<sub>2</sub><br />
<br />
<br><br>&nbsp;&nbsp;&nbsp;&nbsp;On the other hand, light chain takes up Fe(III). By functional expression of these two polypeptides, virtual concentration of Fe inside cell can be much reduced. <br />
Interestingly, the storage capacity, the complex size, and FTH/FTL ratio can vary from species to species. Generally speaking, the mammalian ferritin complex contains more FTL than FTH, while the ferritin complex from bacteria have reversed compositions. <br />
<br><br />
<br><br />
<b>Choice of ferritin</b>: Because the storage capacity of <i>E. coli</i> ferritin is far less than that of mammarian type, we decided to make BioBrick for the functional expression of human ferritin in <i>E. coli</i>.<br />
<br><br />
<br><br />
<b>Hypothesis</b>: By storing Fe in isolation, the maximum capacity for Fe storage should be elevated. Also, the apparent iron tolerance of <i>E. coli</i> should be also elevated. <br />
<br />
</br></br><br />
<br><br />
<p><br />
<br />
<br />
<h3 style="background-color:#ffdead ">2.Experiments</h3><br />
<br />
<p><br />
<h3 style="background-color:#f0ffff ">2.1.BioBrick construction</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;The ratio of FTH/FTL can be flexible in ferritin complex, and there exist a best composition that gives the highest Fe-storage activity. In heterologous expression of ferritin, the translational efficiency can be fine-tuned so that we could achieve that best composition. So, we constructed BioBricks for the functional expression of human ferritin complex in two formats; <br />
<br>BBa_K1057002: 'middle' RBS assigned for FTH <br />
<br>BBa_K1057009: 'strong' RBS assigned for FTH <br />
<br>In both construct, two ferritin genes (FTH and FTL) are placed under pBAD promoter to control the timing and expression level of these genes. To facilitate this construction process, we modified an existing Biobrick (BBa_I74608 deposited by iGEM 2007 team Cambridge) into the new BioBrick(BBa_K1057012). This enabled us the rapid, in-parallel, and one-pot digestion/ ligation using Golden gate method. <br />
<br><br />
<br><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/3/32/Chiba.ferritin.png"alt=""align="middle"></center><br />
<br />
<br />
<center><p><b>Fig. 2</b> Cloning procedure of ferritin-producing BioBrick</p></center><br><br />
<br><br />
</p><br />
<br />
<br />
<a href="https://2013.igem.org/Team:Chiba/Parts"> Parts link</a><br />
</p><br />
<br />
<br />
<h3 style="background-color:#f0ffff ">2.2. Confirmation of Ferritin Expression </h3><br />
<p><br />
Our new BioBricks BBa_K1057002 and BBa_K1057009 were transfected into in BL21 strain. The expression level of each individual components (H-chain and L-chain) was checked by SDS-PAGE/ coomassie blue. As a control, we also conducted the same experiment with “sfgfp generator”.<br />
<br><br />
The results are shown in Fig. 3. Both in total protein fraction (gel in left) and soluble fraction(gel in right), there observed two characteristic bands: one was with the size of 20 kDa (corresonding to FTH), while the other was 19kDa (corresponding to FTL). These bands were not detectable for the control sample (Lane 3) expressing the control gene sfGFP under the control of pBAD promoter.<br />
<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ike.png" width="750px"height="500px"></center><br><br />
<center><p><b>Fig. 3</b> Expression of ferritin in <i>E. coli</i> treated with arabinose. <br />
<br> lane 1. pBAD/araC-ferritin-strong(BBa_K1057009)<br />
<br>lane 2. pBAD/araC-ferritin-mid(BBA_K1057002)<br />
<br>lane 3. BBa_I746908(sfgfp)<br />
</p></center><br />
<br />
<br />
<br />
</p><br />
<br />
<h3 style="background-color:#f0ffff ">2.3.Evaluation of iron tolerance</h3><br />
<p><br />
<br />
<strong>Experiment:</strong> <i>E. coli</i> strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with above plasmids. Here, Human ferritin genes (fth1 and ftl) are under the control of BAD promoter. To express Human ferritin proteins, arabinose (final conc. 0.2%) was added into media. These ferritin generating cell (10^7 cells) inoculated in to fresh media including various concentration of iron citrate (Fe3+) or iron ascorbate (Fe2+). After 12h of incubation (at 37°C), the final cell density and colony forming efficiency was evaluated. As a control, we conducted the same experiment with the cell harboring BBa_I746908 (arabinose-induced sfGFP generator). For the detail of the iron torelance protocol, see <a href="https://2013.igem.org/Team:Chiba/Assay/store">Here. </a><br />
</p><br />
<br />
<h3 style="background-color:#f0ffff ">2.4.Evaluation of magnetism</h3><br />
<p><br />
Now, we finally examined whether BL21 overexpressing ferritin is well attracted to the magnet. Our experimental setup is shown below (Fig. 4). <br> The details of this experimental protocol is given <a href="https://2013.igem.org/Team:Chiba/Assay/store">here</a>.<br />
<center><img src="https://static.igem.org/mediawiki/2013/e/e5/Chiba_magnet_method_ssp.png" width="600px"height="400px"></center><br><br />
<center><p><b>Fig. 4</b> Experimental setup</p></center><br />
<br />
<br><br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid</h3><br />
<br />
<h3 style="background-color:#f0ffff ">3.2 Assessment of iron tolerance</h3><br />
<br><p><b>Result. 1</b><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/6/6e/Chiba_tetu.jpg" width="744px"height="576px"></center><br><br />
<br><center><p><b>Fig. 5</b> Iron tolerance of <i>E. coli</i> harboring ferritin-expressing BioBricks</p></center><br />
<br><br />
<br></p><p><br />
<br>•Over-expression of ferritin didn’t affect the growth so much. <br><br />
•The cell not expressing overexpression of ferritin, growth inhibition was observed when the concentration of ferrous ascorbate is > 6-8 mM. Virtually, no growth was observed ost cells (both BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a>) above that concentration.<br><br />
<br />
•The cell expressing human ferritins showed significantly higher tolerance to the ferrous ascorbate. <br><br />
<br />
•We observed significant difference in viability profiles of the two strains BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a>. In the case of BL21, expression of ferritins improved the cell growth at semi-inhibitory concentration (5-7 mM) of ferrous ascorbate (up to 10 x), but above that concentration, ferritin expression did not rescue the growth of the cell. In case of <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> strain, the final cell density wasn't that elevated, but it rescued the cell growth at higher concentration (7-8 mM) of ferrous ascorbate. We do not know where this difference comes from. It should be noted that <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> is specifically engineered strain so that the cytosolic environment is more oxidizing than the normal E. coli cells. <br><br />
<br />
•The resultant culture was stamped on the fresh agar plate to form colonies. As is shown below (Fig. 5), The higher tolerance of cells expressing ferritin was again obvious: the cell expressing ferritins retains higher viability (colony-forming capacity) than the control. <br />
<br><center><img src="https://static.igem.org/mediawiki/2013/8/86/Chiba_ike_miracle.png" width="716px"></center><br><br />
<br><center><p><b>Fig. 6</b> Colony-forming capability of <i>E. coli</i> with/without ferritin-expressing BioBricks</p></center><br><br />
<br />
<h3 style="background-color:#f0ffff ">3.3 Evaluation of magnetism</h3><br />
<p><b>Result</b><br><br />
Our expectation was very simple; if E. coli is well magnetized, they should be attracted by magnets so that we should see the emergence of a pattern- due to the accumulation of the cell near the magnet.<br />
Alas!, however, we did not see any trends of E. coli flock forming around magnet: nothing really happened....<br />
<br><br>Maybe, we should have done the same experiment in <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> strain where the cytosolic environment is engineered to be more oxidizing. With the same level of iron contents, the ratio of Fe (III), the required state for ferromagnetism), can be significantly improved. Also, the level of iron accumulation was not sufficient at all. Next step we are trying is to further increase in the cellular iron contents by combining this ferritin over expression system with the temporal knocking down of <i>Fur</i> and <i>fie</i>F using CRISPRi technique. </p><br />
<center><img src="https://static.igem.org/mediawiki/2013/9/93/Chiba_tetsu_BL21_str_ssp.png" width="571px"></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/b/b2/Chiba_tetsu_BL21_mid_sp.png" width="571px"></center><br><br />
<p><br />
<center><p><b>Fig. 7</b> Our first attempt to attract <i>E. coli</i> by magnet.</p></center><br />
</p><br />
<br />
<br />
<br />
</div><br />
</body><br />
<br />
</html></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/storeTeam:Chiba/Project/store2013-09-28T02:44:38Z<p>T.Senda: </p>
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<h2 id="storage" style="background-color:#ff9933"><center>Sequestration: Fe-storage machine</center></h2><br />
<br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp; <p><b>Fe must be isolated</b>: In order to magnetize <i>E. coli</i>, we need to stuff as much Fe ions as possible in <i>E. coli</i>. Fe(II) could cause Fenton reaction in response to hydrogen peroxide, harmful hydroxyl radicals (OH•). Dilemma is that, feeding too much Fe into cell would kills the host cell. We decided to over express the ferritins that capture and store Fe irons.</p> <br><br />
<br><br />
<p><b>Fe container machinery</b>: Ferritin particles are made of two small protein subunits (heavy chain (FTH) and light chain (FTL)) (Fig. 1). When expressed, the ferritin subunits automatically assemble into 24-membered protein cages.</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ferrittin.png"alt=""align="middle"></center><br />
<center><p><b>Fig. 1</b> Complex structure of ferritin</p><br></center><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Heavy chain catalyzes oxidation of iron and stimulate 2Fe(II)+O2→[Fe(III)-O-O-Fe(III)] reaction<br />
<br><br />
<br><br />
2Fe<sub>2</sub>+O<sub>2</sub>+(H<sub>2</sub>O)x+3→Fe<sub>2</sub>O<sub>2</sub>(H<sub>2</sub>O)x+4H+H<sub>2</sub>O<sub>2</sub><br />
<br />
<br><br>&nbsp;&nbsp;&nbsp;&nbsp;On the other hand, light chain takes up Fe(III). By functional expression of these two polypeptides, virtual concentration of Fe inside cell can be much reduced. <br />
Interestingly, the storage capacity, the complex size, and FTH/FTL ratio can vary from species to species. Generally speaking, the mammalian ferritin complex contains more FTL than FTH, while the ferritin complex from bacteria have reversed compositions. <br />
<br><br />
<br><br />
<b>Choice of ferritin</b>: Because the storage capacity of <i>E. coli</i> ferritin is far less than that of mammarian type, we decided to make BioBrick for the functional expression of human ferritin in <i>E. coli</i>.<br />
<br><br />
<br><br />
<b>Hypothesis</b>: By storing Fe in isolation, the maximum capacity for Fe storage should be elevated. Also, the apparent iron tolerance of <i>E. coli</i> should be also elevated. <br />
<br />
</br></br><br />
<br><br />
<p><br />
<br />
<br />
<h3 style="background-color:#ffdead ">2.Experiments</h3><br />
<br />
<p><br />
<h3 style="background-color:#f0ffff ">2.1.BioBrick construction</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;The ratio of FTH/FTL can be flexible in ferritin complex, and there exist a best composition that gives the highest Fe-storage activity. In heterologous expression of ferritin, the translational efficiency can be fine-tuned so that we could achieve that best composition. So, we constructed BioBricks for the functional expression of human ferritin complex in two formats; <br />
<br>BBa_K1057002: 'middle' RBS assigned for FTH <br />
<br>BBa_K1057009: 'strong' RBS assigned for FTH <br />
<br>In both construct, two ferritin genes (FTH and FTL) are placed under pBAD promoter to control the timing and expression level of these genes. To facilitate this construction process, we modified an existing Biobrick (BBa_I74608 deposited by iGEM 2007 team Cambridge) into the new BioBrick(BBa_K1057012). This enabled us the rapid, in-parallel, and one-pot digestion/ ligation using Golden gate method. <br />
<br><br />
<br><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/3/32/Chiba.ferritin.png"alt=""align="middle"></center><br />
<br />
<br />
<center><p><b>Fig. 2</b> Cloning procedure of ferritin-producing BioBrick</p></center><br><br />
<br><br />
</p><br />
<br />
<br />
<a href="https://2013.igem.org/Team:Chiba/Parts"> Parts link</a><br />
</p><br />
<br />
<br />
<h3 style="background-color:#f0ffff ">2.2. Confirmation of Ferritin Expression </h3><br />
<p><br />
Our new BioBricks BBa_K1057002 and BBa_K1057009 were transfected into in BL21 strain. The expression level of each individual components (H-chain and L-chain) was checked by SDS-PAGE/ coomassie blue. As a control, we also conducted the same experiment with “sfgfp generator”.<br />
<br><br />
The results are shown in Fig. 3. Both in total protein fraction (gel in left) and soluble fraction(gel in right), there observed two characteristic bands: one was with the size of 20 kDa (corresonding to FTH), while the other was 19kDa (corresponding to FTL). These bands were not detectable for the control sample (Lane 3) expressing the control gene sfGFP under the control of pBAD promoter.<br />
<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ike.png" width="750px"height="500px"></center><br><br />
<center><p><b>Fig. 3</b> Expression of ferritin in <i>E. coli</i> treated with arabinose. <br />
<br> lane 1. pBAD/araC-ferritin-strong(BBa_K1057009)<br />
<br>lane 2. pBAD/araC-ferritin-mid(BBA_K1057002)<br />
<br>lane 3. BBa_I746908(sfgfp)<br />
</p></center><br />
<br />
<br />
<br />
</p><br />
<br />
<h3 style="background-color:#f0ffff ">2.3.Evaluation of iron tolerance</h3><br />
<p><br />
<br />
<strong>Experiment:</strong> <i>E. coli</i> strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with above plasmids. Here, Human ferritin genes (fth1 and ftl) are under the control of BAD promoter. To express Human ferritin proteins, arabinose (final conc. 0.2%) was added into media. These ferritin generating cell (10^7 cells) inoculated in to fresh media including various concentration of iron citrate (Fe3+) or iron ascorbate (Fe2+). After 12h of incubation (at 37°C), the final cell density and colony forming efficiency was evaluated. As a control, we conducted the same experiment with the cell harboring BBa_I746908 (arabinose-induced sfGFP generator). For the detail of the iron torelance protocol, see <a href="https://2013.igem.org/Team:Chiba/Assay/store">Here. </a><br />
</p><br />
<br />
<h3 style="background-color:#f0ffff ">2.4.Evaluation of magnetism</h3><br />
<p><br />
Now, we finally examined whether BL21 overexpressing ferritin is well attracted to the magnet. Our experimental setup is shown below (Fig. 4). <br> The details of this experimental protocol is given <a href="https://2013.igem.org/Team:Chiba/Assay/store">here</a>.<br />
<center><img src="https://static.igem.org/mediawiki/2013/e/e5/Chiba_magnet_method_ssp.png" width="600px"height="400px"></center><br><br />
<center><p><b>Fig. 4</b> Experimental setup</p></center><br />
<br />
<br><br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid</h3><br />
<br />
<h3 style="background-color:#f0ffff ">3.2 Assessment of iron tolerance</h3><br />
<br><p><b>Result. 1</b><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/6/6e/Chiba_tetu.jpg" width="744px"height="576px"></center><br><br />
<br><center><p><b>Fig. 5</b> Iron tolerance of E. coli harboring ferritin-expressing BioBricks</p></center><br />
<br><br />
<br></p><p><br />
<br>•Over-expression of ferritin didn’t affect the growth so much. <br><br />
•The cell not expressing overexpression of ferritin, growth inhibition was observed when the concentration of ferrous ascorbate is > 6-8 mM. Virtually, no growth was observed ost cells (both BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a>) above that concentration.<br><br />
<br />
•The cell expressing human ferritins showed significantly higher tolerance to the ferrous ascorbate. <br><br />
<br />
•We observed significant difference in viability profiles of the two strains BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a>. In the case of BL21, expression of ferritins improved the cell growth at semi-inhibitory concentration (5-7 mM) of ferrous ascorbate (up to 10 x), but above that concentration, ferritin expression did not rescue the growth of the cell. In case of <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> strain, the final cell density wasn't that elevated, but it rescued the cell growth at higher concentration (7-8 mM) of ferrous ascorbate. We do not know where this difference comes from. It should be noted that <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> is specifically engineered strain so that the cytosolic environment is more oxidizing than the normal E. coli cells. <br><br />
<br />
•The resultant culture was stamped on the fresh agar plate to form colonies. As is shown below (Fig. 5), The higher tolerance of cells expressing ferritin was again obvious: the cell expressing ferritins retains higher viability (colony-forming capacity) than the control. <br />
<br><center><img src="https://static.igem.org/mediawiki/2013/8/86/Chiba_ike_miracle.png" width="716px"></center><br><br />
<br><center><p><b>Fig. 6</b> Colony-forming capability of <i>E. coli</i> with/without ferritin-expressing BioBricks</p></center><br><br />
<br />
<h3 style="background-color:#f0ffff ">3.3 Evaluation of magnetism</h3><br />
<p><b>Result</b><br><br />
Our expectation was very simple; if E. coli is well magnetized, they should be attracted by magnets so that we should see the emergence of a pattern- due to the accumulation of the cell near the magnet.<br />
Alas!, however, we did not see any trends of E. coli flock forming around magnet: nothing really happened....<br />
<br><br>Maybe, we should have done the same experiment in <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> strain where the cytosolic environment is engineered to be more oxidizing. With the same level of iron contents, the ratio of Fe (III), the required state for ferromagnetism), can be significantly improved. Also, the level of iron accumulation was not sufficient at all. Next step we are trying is to further increase in the cellular iron contents by combining this ferritin over expression system with the temporal knocking down of <i>Fur</i> and <i>fie</i>F using CRISPRi technique. </p><br />
<center><img src="https://static.igem.org/mediawiki/2013/9/93/Chiba_tetsu_BL21_str_ssp.png" width="571px"></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/b/b2/Chiba_tetsu_BL21_mid_sp.png" width="571px"></center><br><br />
<p><br />
<center><p><b>Fig. 7</b> Our first attempt to attract E. coli by magnet.</p></center><br />
</p><br />
<br />
<br />
<br />
</div><br />
</body><br />
<br />
</html></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/storeTeam:Chiba/Project/store2013-09-28T02:40:04Z<p>T.Senda: </p>
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<head><br />
<meta http-equiv="Content-Type" content="text/html;charset=utf-8"/><br />
<title>iGEM-2013 Chiba</title><br />
<br />
<style type="text/css"><br />
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</head><br />
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<h2 id="storage" style="background-color:#ff9933"><center>Sequestration: Fe-storage machine</center></h2><br />
<br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp; <p><b>Fe must be isolated</b>: In order to magnetize <i>E. coli</i>, we need to stuff as much Fe ions as possible in <i>E. coli</i>. Fe(II) could cause Fenton reaction in response to hydrogen peroxide, harmful hydroxyl radicals (OH•). Dilemma is that, feeding too much Fe into cell would kills the host cell. We decided to over express the ferritins that capture and store Fe irons.</p> <br><br />
<br><br />
<p><b>Fe container machinery</b>: Ferritin particles are made of two small protein subunits (heavy chain (FTH) and light chain (FTL)) (Fig. 1). When expressed, the ferritin subunits automatically assemble into 24-membered protein cages.</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ferrittin.png"alt=""align="middle"></center><br />
<center><p><b>Fig. 1</b> Complex structure of ferritin</p><br></center><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Heavy chain catalyzes oxidation of iron and stimulate 2Fe(II)+O2→[Fe(III)-O-O-Fe(III)] reaction<br />
<br><br />
<br><br />
2Fe<sub>2</sub>+O<sub>2</sub>+(H<sub>2</sub>O)x+3→Fe<sub>2</sub>O<sub>2</sub>(H<sub>2</sub>O)x+4H+H<sub>2</sub>O<sub>2</sub><br />
<br />
<br><br>&nbsp;&nbsp;&nbsp;&nbsp;On the other hand, light chain takes up Fe(III). By functional expression of these two polypeptides, virtual concentration of Fe inside cell can be much reduced. <br />
Interestingly, the storage capacity, the complex size, and FTH/FTL ratio can vary from species to species. Generally speaking, the mammalian ferritin complex contains more FTL than FTH, while the ferritin complex from bacteria have reversed compositions. <br />
<br><br />
<br><br />
<b>Choice of ferritin</b>: Because the storage capacity of <i>E. coli</i> ferritin is far less than that of mammarian type, we decided to make BioBrick for the functional expression of human ferritin in <i>E. coli</i>.<br />
<br><br />
<br><br />
<b>Hypothesis</b>: By storing Fe in isolation, the maximum capacity for Fe storage should be elevated. Also, the apparent iron tolerance of <i>E. coli</i> should be also elevated. <br />
<br />
</br></br><br />
<br><br />
<p><br />
<br />
<br />
<h3 style="background-color:#ffdead ">2.Experiments</h3><br />
<br />
<p><br />
<h3 style="background-color:#f0ffff ">2.1.BioBrick construction</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;The ratio of FTH/FTL can be flexible in ferritin complex, and there exist a best composition that gives the highest Fe-storage activity. In heterologous expression of ferritin, the translational efficiency can be fine-tuned so that we could achieve that best composition. So, we constructed BioBricks for the functional expression of human ferritin complex in two formats; <br />
<br>BBa_K1057002: 'middle' RBS assigned for FTH <br />
<br>BBa_K1057009: 'strong' RBS assigned for FTH <br />
<br>In both construct, two ferritin genes (FTH and FTL) are placed under pBAD promoter to control the timing and expression level of these genes. To facilitate this construction process, we modified an existing Biobrick (BBa_I74608 deposited by iGEM 2007 team Cambridge) into the new BioBrick(BBa_K1057012). This enabled us the rapid, in-parallel, and one-pot digestion/ ligation using Golden gate method. <br />
<br><br />
<br><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/3/32/Chiba.ferritin.png"alt=""align="middle"></center><br />
<br />
<br />
<center><p><b>Fig. 2</b> Cloning procedure of ferritin-producing BioBrick</p></center><br><br />
<br><br />
</p><br />
<br />
<br />
<a href="https://2013.igem.org/Team:Chiba/Parts"> Parts link</a><br />
</p><br />
<br />
<br />
<h3 style="background-color:#f0ffff ">2.2. Confirmation of Ferritin Expression </h3><br />
<p><br />
Our new BioBricks BBa_K1057002 and BBa_K1057009 were transfected into in BL21 strain. The expression level of each individual components (H-chain and L-chain) was checked by SDS-PAGE/ coomassie blue. As a control, we also conducted the same experiment with “sfgfp generator”.<br />
<br><br />
The results are shown in Fig. 3. Both in total protein fraction (gel in left) and soluble fraction(gel in right), there observed two characteristic bands: one was with the size of 20 kDa (corresonding to FTH), while the other was 19kDa (corresponding to FTL). These bands were not detectable for the control sample (Lane 3) expressing the control gene sfGFP under the control of pBAD promoter.<br />
<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ike.png" width="750px"height="500px"></center><br><br />
<center><p><b>Fig. 3</b> Expression of ferritin in <i>E. coli</i> treated with arabinose. <br />
<br> lane 1. pBAD/araC-ferritin-strong(BBa_K1057009)<br />
<br>lane 2. pBAD/araC-ferritin-mid(BBA_K1057002)<br />
<br>lane 3. BBa_I746908(sfgfp)<br />
</p></center><br />
<br />
<br />
<br />
</p><br />
<br />
<h3 style="background-color:#f0ffff ">2.3.Evaluation of iron tolerance</h3><br />
<p><br />
<br />
<strong>Experiment:</strong> E. coli strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with above plasmids. Here, Human ferritin genes (fth1 and ftl) are under the control of BAD promoter. To express Human ferritin proteins, arabinose (final conc. 0.2%) was added into media. These ferritin generating cell (10^7 cells) inoculated in to fresh media including various concentration of iron citrate (Fe3+) or iron ascorbate (Fe2+). After 12h of incubation (at 37°C), the final cell density and colony forming efficiency was evaluated. As a control, we conducted the same experiment with the cell harboring BBa_I746908 (arabinose-induced sfGFP generator). For the detail of the iron torelance protocol, see <a href="https://2013.igem.org/Team:Chiba/Assay/store">Here. </a><br />
</p><br />
<br />
<h3 style="background-color:#f0ffff ">2.4.Evaluation of magnetism</h3><br />
<p><br />
Now, we finally examined whether BL21 overexpressing ferritin is well attracted to the magnet. Our experimental setup is shown below (Fig. 4). <br> The details of this experimental protocol is given <a href="https://2013.igem.org/Team:Chiba/Assay/store">here</a>.<br />
<center><img src="https://static.igem.org/mediawiki/2013/e/e5/Chiba_magnet_method_ssp.png" width="600px"height="400px"></center><br><br />
<center><p><b>Fig. 4</b> Experimental setup</p></center><br />
<br />
<br><br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid</h3><br />
<br />
<h3 style="background-color:#f0ffff ">3.2 Assessment of iron tolerance</h3><br />
<br><p><b>Result. 1</b><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/6/6e/Chiba_tetu.jpg" width="744px"height="576px"></center><br><br />
<br><center><p><b>Fig. 5</b> Iron tolerance of E. coli harboring ferritin-expressing BioBricks</p></center><br />
<br><br />
<br></p><p><br />
<br>•Over-expression of ferritin didn’t affect the growth so much. <br><br />
•The cell not expressing overexpression of ferritin, growth inhibition was observed when the concentration of ferrous ascorbate is > 6-8 mM. Virtually, no growth was observed ost cells (both BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a>) above that concentration.<br><br />
<br />
•The cell expressing human ferritins showed significantly higher tolerance to the ferrous ascorbate. <br><br />
<br />
•We observed significant difference in viability profiles of the two strains BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a>. In the case of BL21, expression of ferritins improved the cell growth at semi-inhibitory concentration (5-7 mM) of ferrous ascorbate (up to 10 x), but above that concentration, ferritin expression did not rescue the growth of the cell. In case of <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> strain, the final cell density wasn't that elevated, but it rescued the cell growth at higher concentration (7-8 mM) of ferrous ascorbate. We do not know where this difference comes from. It should be noted that <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> is specifically engineered strain so that the cytosolic environment is more oxidizing than the normal E. coli cells. <br><br />
<br />
•The resultant culture was stamped on the fresh agar plate to form colonies. As is shown below (Fig. 5), The higher tolerance of cells expressing ferritin was again obvious: the cell expressing ferritins retains higher viability (colony-forming capacity) than the control. <br />
<br><center><img src="https://static.igem.org/mediawiki/2013/8/86/Chiba_ike_miracle.png" width="716px"height="299px"></center><br><br />
<br><center><p><b>Fig. 6</b> Colony-forming capability of E. coli with/without ferritin-expressing BioBricks</p></center><br><br />
<br />
<h3 style="background-color:#f0ffff ">3.3 Evaluation of magnetism</h3><br />
<p><b>Result</b><br><br />
Our expectation was very simple; if E. coli is well magnetized, they should be attracted by magnets so that we should see the emergence of a pattern- due to the accumulation of the cell near the magnet.<br />
Alas!, however, we did not see any trends of E. coli flock forming around magnet: nothing really happened....<br />
<br><br>Maybe, we should have done the same experiment in <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> strain where the cytosolic environment is engineered to be more oxidizing. With the same level of iron contents, the ratio of Fe (III), the required state for ferromagnetism), can be significantly improved. Also, the level of iron accumulation was not sufficient at all. Next step we are trying is to further increase in the cellular iron contents by combining this ferritin over expression system with the temporal knocking down of <i>Fur</i> and <i>fie</i>F using CRISPRi technique. </p><br />
<center><img src="https://static.igem.org/mediawiki/2013/9/93/Chiba_tetsu_BL21_str_ssp.png" width="571px"></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/b/b2/Chiba_tetsu_BL21_mid_sp.png" width="571px"></center><br><br />
<p><br />
<center><p><b>Fig. 7</b> Our first attempt to attract E. coli by magnet.</p></center><br />
</p><br />
<br />
<br />
<br />
</div><br />
</body><br />
<br />
</html></div>T.Sendahttp://2013.igem.org/File:Chiba_ike_miracle.pngFile:Chiba ike miracle.png2013-09-28T02:32:52Z<p>T.Senda: </p>
<hr />
<div></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/uptakeTeam:Chiba/Project/uptake2013-09-28T02:03:27Z<p>T.Senda: </p>
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<h2 id="uptake" style="background-color:#ff9933"><center>Iron Uptake</center></h2><br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<br />
<p><br />
<h3 style="background-color:#f0ffff ">1.1.<i>Fur</i></h3><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;<i>Fur</i> (Ferric uptake regulator) controls iron metabolism such as taking iron in or storing iron.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In most cases, it combines with <i>Fur</i> box (which is near the promoter) and regulates transcription.</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;When iron is rich, <i>Fur</i> becomes active and when <i>Fur</i> becomes active, it restricts the expression of iron transporter, and that means that the iron uptake would stop. At the same time,<i> Fur</i> restricts the expression of <i>Ryh</i>B. <i>RyhB</i> is one of the sRNA that restricts the expression of Ferritin, so making <i>Fur</i> active leads ferritin to express.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Conversely, when iron is lacking, <i>Fur</i> becomes inactive, and the iron transporter would work normally, so iron would be taken in. <i>Ryh</i>B would also work normally, so expression of ferritin is restricted and Ferritin wouldn't be expressed.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In short, if we knockdown/knockout <i>Fur</i>, iron transporter would be active so iron would be taken in, but the expression of Ferritin is stopped by <i>Ryh</i>B.</p><br><br />
<br><br />
<h3 style="background-color:#f0ffff ">1.2.<i>fie</i>F</h3><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;There is a Regulator called <i>fie</i>F (ferric iron efflux). It makes iron and zinc flow out of cell and controls detoxification of cell. When <i>fie</i>F is knocked down/out, the tolerance of cell to iron would be lowered.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;<i>Fec</i> has a character that can recognize ferric citrate and taking Fe(III) in. <i>Fec</i> is controlled by <i>Fur</i> and when the density of Fe(III) in the cell is too high, <i>Fur</i> restricts Fec and stops iron uptake.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In the same way, <i>Efe</i>UOB has a character that can recognize ascorbic acid and taking Fe(II) in. <i>Efe</i>UOB is also controlled by <i>Fur</i> and when the density of Fe(II) in the cell is too high, <i>Fur</i> restricts <i>EfeUOB</i> and stops iron uptake.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Using these characters, and by knocking down/out <i>Fur</i> and <i>fie</i>F, <i>Fec</i>/<i>Efe</i>UOB expression wouldn't be restricted so the amount of iron coming in would increase and the amount of iron going out would decrease. The system would work like when iron is lacking.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;As a result, the amount of iron inside <i>E. coli</i> would increase.</p><br />
<br><center><img src="https://static.igem.org/mediawiki/2013/f/f8/Uptake_image.PNG" width="723px"height="305px"></center><br><br />
<br />
</p><br />
<br />
<br />
<h3 style="background-color:#ffdead ">2.Materials&Methods</h3><br />
<br />
<h3 style="background-color:#f0ffff ">2.1.plasmid construct</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We constructed four plasmids knocking down <i>fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. at the same time, in order to confirm the function of dCas9, we constructed a plasmid knocking down <i>lacZ</i>.<br><br />
<br><br />
<br />
<br />
<br />
<a href="#">Part link</a><br><br><br />
</p><br />
<h3 style="background-color:#f0ffff ">2.2.Evaluation of <i>Fur</i>,<i>fie</i>F knockdown</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We performed two experiments about CRISPRi system in order to confirm the knockdown function as desired. The purpose is a function check for <i>lacZ</i>, <i>Fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. <br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/7/7f/Chiba.CRISPRi.kan.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 1</b> the method of function check about <i>fur</i></p></center><br />
<br />
<br />
<br />
<a href="https://2013.igem.org/Team:Chiba/Assay/uptake">Assay</a><br />
</p><br />
<h3 style="background-color:#f0ffff ">2.3.Evaluation of absorbed iron volume</h3><br />
<p><br />
<b>Experiment:</b><br><br />
<br />
<br />
<left><img src="https://static.igem.org/mediawiki/2013/3/3f/CRISPRi.PNG" width="409px"height="88px"></left><br />
<right><img src="https://static.igem.org/mediawiki/2013/2/23/DCas9.PNG" width="306px"height="110px"></right><br><br />
<center><b>Fig. a</b></center><br />
<br><p>&nbsp;&nbsp;&nbsp;&nbsp;<i>E. coli</i> stain BL21 was transformed by Plasmid shown in <b>Fig. a</b> .&nbsp;Then we cultured all transformants with atC.&nbsp; atC was added to knock down <i>fur</i> and <i>fie</i>F.&nbsp; After that we cultured it in the presence of ferric citrate, and measured the density of iron that weren’t taken in to <i>E. coli</i> and remained in the medium by measuring Absorbance (Abs 756) with color reagent.</p><br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<p><br />
<br />
<br />
<h3 style="background-color:#f0ffff ">3.1.Function Check</h3><br />
<p><br />
1) In the absence of anhydro Tetracycline (aTc), CRISPRi-medeated <i>lac</i>Z gene transcriptional silencing did not occur, resulting in blue-colored colony in which <i>lac</i>Z was expressed.<br><br />
2) In the presence of aTc, CRISPRi-medeated <i>lac</i>Z gene transcriptional silencing successfully occurred, resulting in colorless colony in which <i>lac</i>Z was not expressed.<br><br />
3) CRISPRi-medeated <i>fur</i> gene transcriptional silencing successfully occurred, resulting in the loss of kanamysin resistance.<br><br />
</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/26/Chiba.CRISPRi.spot.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 2</b>CRISPRi efficiently silence transcription</p></center><br><br />
<br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/a/a6/Chiba.CRISPRi-lacZ.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 3</b> Function check about CRISPRi-<i>lacZ</i></p></center><br><br />
<br />
<br />
<br><br />
<br />
<br />
<br />
<br />
<h3 style="background-color:#f0ffff ">3.2.A knockdown of <i>fie</i>F or fur has no effect on iron uptake </h3><br />
<br />
<p><br />
Iron uptake was not detectable (iron concentration changes in media were less than micro molar order), even if <i>fur</i> or <i>fie</i>F was successfully knocked down.<br><br />
A future subject is to experiment at more cell number of <i>E. coli</i>.<br><br />
</p><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/0/0d/Chiba_shimamura_super.png"width="800px"></center><br><br />
<center><p><b>Fig. 4 </b>Absorbance at each cell number of E. coli(BL21 and SHuffle® introduced each plusmid</p></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/5/5e/Chiba_shimamura_kennryousen.png"width="800px"></center><br><br />
<center><p><b>Fig. 5</b> Absorbance of as a function of each iron concentration</p></center><br><br />
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<br />
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</html></div>T.Sendahttp://2013.igem.org/File:Chiba_shimamura_super.pngFile:Chiba shimamura super.png2013-09-28T02:01:40Z<p>T.Senda: </p>
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<div></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/uptakeTeam:Chiba/Project/uptake2013-09-28T01:40:32Z<p>T.Senda: </p>
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<title>iGEM-2013 Chiba</title><br />
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<style type="text/css"><br />
<br />
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<h2 id="uptake" style="background-color:#ff9933"><center>Uptake</center></h2><br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<br />
<p><br />
<h3 style="background-color:#f0ffff ">1.1.<i>Fur</i></h3><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;<i>Fur</i> (Ferric uptake regulator) controls iron metabolism such as taking iron in or storing iron.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In most cases, it combines with <i>Fur</i> box (which is near the promoter) and regulates transcription.</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;When iron is rich, <i>Fur</i> becomes active and when <i>Fur</i> becomes active, it restricts the expression of iron transporter, and that means that the iron uptake would stop. At the same time,<i> Fur</i> restricts the expression of <i>Ryh</i>B. <i>RyhB</i> is one of the sRNA that restricts the expression of Ferritin, so making <i>Fur</i> active leads ferritin to express.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Conversely, when iron is lacking, <i>Fur</i> becomes inactive, and the iron transporter would work normally, so iron would be taken in. <i>Ryh</i>B would also work normally, so expression of ferritin is restricted and Ferritin wouldn't be expressed.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In short, if we knockdown/knockout <i>Fur</i>, iron transporter would be active so iron would be taken in, but the expression of Ferritin is stopped by <i>Ryh</i>B.</p><br><br />
<br><br />
<h3 style="background-color:#f0ffff ">1.2.<i>fie</i>F</h3><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;There is a Regulator called <i>fie</i>F (ferric iron efflux). It makes iron and zinc flow out of cell and controls detoxification of cell. When <i>fie</i>F is knocked down/out, the tolerance of cell to iron would be lowered.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;<i>Fec</i> has a character that can recognize ferric citrate and taking Fe(III) in. <i>Fec</i> is controlled by <i>Fur</i> and when the density of Fe(III) in the cell is too high, <i>Fur</i> restricts Fec and stops iron uptake.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In the same way, <i>Efe</i>UOB has a character that can recognize ascorbic acid and taking Fe(II) in. <i>Efe</i>UOB is also controlled by <i>Fur</i> and when the density of Fe(II) in the cell is too high, <i>Fur</i> restricts <i>EfeUOB</i> and stops iron uptake.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Using these characters, and by knocking down/out <i>Fur</i> and <i>fie</i>F, <i>Fec</i>/<i>Efe</i>UOB expression wouldn't be restricted so the amount of iron coming in would increase and the amount of iron going out would decrease. The system would work like when iron is lacking.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;As a result, the amount of iron inside <i>E. coli</i> would increase.</p><br />
<br><center><img src="https://static.igem.org/mediawiki/2013/f/f8/Uptake_image.PNG" width="723px"height="305px"></center><br><br />
<br />
</p><br />
<br />
<br />
<h3 style="background-color:#ffdead ">2.Materials&Methods</h3><br />
<br />
<h3 style="background-color:#f0ffff ">2.1.plasmid construct</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We constructed four plasmids knocking down <i>fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. at the same time, in order to confirm the function of dCas9, we constructed a plasmid knocking down <i>lacZ</i>.<br><br />
プラスミドについて書く<br><br />
<br />
<br />
<br />
<a href="#">Part link</a><br><br><br />
</p><br />
<h3 style="background-color:#f0ffff ">2.2.Evaluation of <i>Fur</i>,<i>fie</i>F knockdown</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We performed two experiments about CRISPRi system in order to confirm the knockdown function as desired. The purpose is a function check for <i>lacZ</i>, <i>Fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. <br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/7/7f/Chiba.CRISPRi.kan.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 1</b> the method of function check about <i>fur</i></p></center><br />
<br />
<br />
<br />
<a href="https://2013.igem.org/Team:Chiba/Assay/uptake">Assay</a><br />
</p><br />
<h3 style="background-color:#f0ffff ">2.3.Evaluation of absorbed iron volume</h3><br />
<p><br />
<b>Experiment:</b><br><br />
<br />
<br />
<left><img src="https://static.igem.org/mediawiki/2013/3/3f/CRISPRi.PNG" width="409px"height="88px"></left><br />
<right><img src="https://static.igem.org/mediawiki/2013/2/23/DCas9.PNG" width="306px"height="110px"></right><br><br />
<center><b>Fig. a</b></center><br />
<br><p>&nbsp;&nbsp;&nbsp;&nbsp;<i>E. coli</i> stain BL21 was transformed by Plasmid shown in <b>Fig. a</b> .&nbsp;Then we cultured all transformants with atC.&nbsp; atC was added to knock down <i>fur</i> and <i>fie</i>F.&nbsp; After that we cultured it in the presence of ferric citrate, and measured the density of iron that weren’t taken in to <i>E. coli</i> and remained in the medium by measuring Absorbance (Abs 756) with color reagent.</p><br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<p><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid建設の説明</h3><br />
<br />
<h3 style="background-color:#f0ffff ">3.2.Function Check</h3><br />
<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/26/Chiba.CRISPRi.spot.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 2</b>CRISPRi efficiently silence transcription</p></center><br><br />
<br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/a/a6/Chiba.CRISPRi-lacZ.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 3</b> Function check about CRISPRi-<i>lacZ</i></p></center><br><br />
<br />
1) In the absence of anhydro Tetracycline (aTc), CRISPRi-medeated <i>lac</i>Z gene transcriptional silencing did not occur, resulting in blue-colored colony in which <i>lac</i>Z was expressed.<br><br />
2) In the presence of aTc, CRISPRi-medeated <i>lac</i>Z gene transcriptional silencing successfully occurred, resulting in colorless colony in which <i>lac</i>Z was not expressed.<br><br />
3) CRISPRi-medeated <i>fur</i> gene transcriptional silencing successfully occurred, resulting in the loss of kanamysin resistance.<br><br />
<br />
<br />
<br><br />
<br />
<br />
<br />
<br />
<h3 style="background-color:#f0ffff ">3.3.A knockdown of <i>fie</i>F or fur has no effect on iron uptake </h3><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/f/f0/Chiba_shimamura_special.png"width="800px"></center><br><br />
<center>Fig. 4 Absorbance at each cell number of E. coli(BL21 and SHuffle® introduced each plusmid</center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/5/5e/Chiba_shimamura_kennryousen.png"width="800px"></center><br><br />
<center>Fig. 5 Absorbance of as a function of each iron concentration</center><br><br />
<br />
<br />
Iron uptake was not detectable (iron concentration changes in media were less than micro molar order), even if <i>fur</i> or <i>fie</i>F was successfully knocked down.<br><br />
A future subject is to experiment at more cell number of <i>E. coli</i>.<br><br />
<br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</body><br />
<br />
</html></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/uptakeTeam:Chiba/Project/uptake2013-09-28T01:36:34Z<p>T.Senda: </p>
<hr />
<div>{{Chiba_base}}<br />
<br />
<html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html;charset=utf-8"/><br />
<title>iGEM-2013 Chiba</title><br />
<br />
<style type="text/css"><br />
<br />
<br />
<br />
</style><br />
<br />
</head><br />
<br />
<body><br />
<div class="textSpace"><br />
<br />
<br />
<h2 id="uptake" style="background-color:#ff9933"><center>Uptake</center></h2><br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<br />
<p><br />
<h3 style="background-color:#f0ffff ">1.1.<i>Fur</i></h3><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;<i>Fur</i> (Ferric uptake regulator) controls iron metabolism such as taking iron in or storing iron.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In most cases, it combines with <i>Fur</i> box (which is near the promoter) and regulates transcription.</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;When iron is rich, <i>Fur</i> becomes active and when <i>Fur</i> becomes active, it restricts the expression of iron transporter, and that means that the iron uptake would stop. At the same time,<i> Fur</i> restricts the expression of <i>Ryh</i>B. <i>RyhB</i> is one of the sRNA that restricts the expression of Ferritin, so making <i>Fur</i> active leads ferritin to express.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Conversely, when iron is lacking, <i>Fur</i> becomes inactive, and the iron transporter would work normally, so iron would be taken in. <i>Ryh</i>B would also work normally, so expression of ferritin is restricted and Ferritin wouldn't be expressed.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In short, if we knockdown/knockout <i>Fur</i>, iron transporter would be active so iron would be taken in, but the expression of Ferritin is stopped by <i>Ryh</i>B.</p><br><br />
<br><br />
<h3 style="background-color:#f0ffff ">1.2.<i>fie</i>F</h3><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;There is a Regulator called <i>fie</i>F (ferric iron efflux). It makes iron and zinc flow out of cell and controls detoxification of cell. When <i>fie</i>F is knocked down/out, the tolerance of cell to iron would be lowered.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;<i>Fec</i> has a character that can recognize ferric citrate and taking Fe(III) in. <i>Fec</i> is controlled by <i>Fur</i> and when the density of Fe(III) in the cell is too high, <i>Fur</i> restricts Fec and stops iron uptake.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In the same way, <i>Efe</i>UOB has a character that can recognize ascorbic acid and taking Fe(II) in. <i>Efe</i>UOB is also controlled by <i>Fur</i> and when the density of Fe(II) in the cell is too high, <i>Fur</i> restricts <i>EfeUOB</i> and stops iron uptake.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Using these characters, and by knocking down/out <i>Fur</i> and <i>fie</i>F, <i>Fec</i>/<i>Efe</i>UOB expression wouldn't be restricted so the amount of iron coming in would increase and the amount of iron going out would decrease. The system would work like when iron is lacking.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;As a result, the amount of iron inside <i>E. coli</i> would increase.</p><br />
<br><center><img src="https://static.igem.org/mediawiki/2013/f/f8/Uptake_image.PNG" width="723px"height="305px"></center><br><br />
<br />
</p><br />
<br />
<br />
<h3 style="background-color:#ffdead ">2.Materials&Methods</h3><br />
<br />
<h3 style="background-color:#f0ffff ">2.1.plasmid construct</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We constructed four plasmids knocking down <i>fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. at the same time, in order to confirm the function of dCas9, we constructed a plasmid knocking down <i>lacZ</i>.<br><br />
プラスミドについて書く<br><br />
<br />
<br />
<br />
<a href="#">Part link</a><br><br><br />
</p><br />
<h3 style="background-color:#f0ffff ">2.2.Evaluation of <i>Fur</i>,<i>fie</i>F knockdown</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We performed two experiments about CRISPRi system in order to confirm the knockdown function as desired. The purpose is a function check for <i>lacZ</i>, <i>Fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. <br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/7/7f/Chiba.CRISPRi.kan.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 1</b> the method of function check about <i>fur</i></p></center><br />
<br />
<br />
<br />
<a href="https://2013.igem.org/Team:Chiba/Assay/uptake">Assay</a><br />
</p><br />
<h3 style="background-color:#f0ffff ">2.3.Evaluation of absorbed iron volume</h3><br />
<p><br />
<b>Experiment:</b><br><br />
<br />
<br />
<left><img src="https://static.igem.org/mediawiki/2013/3/3f/CRISPRi.PNG" width="409px"height="88px"></left><br />
<right><img src="https://static.igem.org/mediawiki/2013/2/23/DCas9.PNG" width="306px"height="110px"></right><br><br />
<center><b>Fig. a</b></center><br />
<br><p>&nbsp;&nbsp;&nbsp;&nbsp;<i>E. coli</i> stain BL21 was transformed by Plasmid shown in <b>Fig. a</b> .&nbsp;Then we cultured all transformants with atC.&nbsp; atC was added to knock down <i>fur</i> and <i>fie</i>F.&nbsp; After that we cultured it in the presence of ferric citrate, and measured the density of iron that weren’t taken in to <i>E. coli</i> and remained in the medium by measuring Absorbance (Abs 756) with color reagent.</p><br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<p><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid建設の説明</h3><br />
<br />
<h3 style="background-color:#f0ffff ">3.2.Function Check</h3><br />
<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/26/Chiba.CRISPRi.spot.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 2</b>CRISPRi efficiently silence transcription</p></center><br><br />
<br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/a/a6/Chiba.CRISPRi-lacZ.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 3</b> Function check about CRISPRi-<i>lacZ</i></p></center><br><br />
<br />
1) In the absence of anhydro Tetracycline (aTc), CRISPRi-medeated <i>lac</i>Z gene transcriptional silencing did not occur, resulting in blue-colored colony in which <i>lac</i>Z was expressed.<br><br />
2) In the presence of aTc, CRISPRi-medeated <i>lac</i>Z gene transcriptional silencing successfully occurred, resulting in colorless colony in which <i>lac</i>Z was not expressed.<br><br />
3) CRISPRi-medeated <i>fur</i> gene transcriptional silencing successfully occurred, resulting in the loss of kanamysin resistance.<br><br />
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<br><br />
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<h3 style="background-color:#f0ffff ">3.3.A knockdown of <i>fie</i>F or fur has no effect on iron uptake </h3><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/f/f0/Chiba_shimamura_special.png"width="800px"></center><br><br />
<center>Fig. 4 Absorbance at each cell number of E. coli(BL21 and SHuffle® introduced each plusmid</center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/5/5e/Chiba_shimamura_kennryousen.png"width="800px"></center><br><br />
<center>Fig. 5 Absorbance of as a function of each iron concentration</center><br><br />
<br />
<br />
Iron uptake was not detectable (iron concentration changes in media were less than micro molar order), even if <i>fur</i> or <i>fie</i>F was successfully knocked down.<br><br />
A future subject is to experiment <br />
今後の課題としては、さらに多い細胞数での定量、または、加える鉄試薬の濃度をさらに調整しなおす必要がある。<br><br />
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<h2 id="uptake" style="background-color:#ff9933"><center>Uptake</center></h2><br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
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<p><br />
<h3 style="background-color:#f0ffff ">1.1.<i>Fur</i></h3><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;<i>Fur</i> (Ferric uptake regulator) controls iron metabolism such as taking iron in or storing iron.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In most cases, it combines with <i>Fur</i> box (which is near the promoter) and regulates transcription.</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;When iron is rich, <i>Fur</i> becomes active and when <i>Fur</i> becomes active, it restricts the expression of iron transporter, and that means that the iron uptake would stop. At the same time,<i> Fur</i> restricts the expression of <i>Ryh</i>B. <i>RyhB</i> is one of the sRNA that restricts the expression of Ferritin, so making <i>Fur</i> active leads ferritin to express.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Conversely, when iron is lacking, <i>Fur</i> becomes inactive, and the iron transporter would work normally, so iron would be taken in. <i>Ryh</i>B would also work normally, so expression of ferritin is restricted and Ferritin wouldn't be expressed.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In short, if we knockdown/knockout <i>Fur</i>, iron transporter would be active so iron would be taken in, but the expression of Ferritin is stopped by <i>Ryh</i>B.</p><br><br />
<br><br />
<h3 style="background-color:#f0ffff ">1.2.<i>fie</i>F</h3><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;There is a Regulator called <i>fie</i>F (ferric iron efflux). It makes iron and zinc flow out of cell and controls detoxification of cell. When <i>fie</i>F is knocked down/out, the tolerance of cell to iron would be lowered.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;<i>Fec</i> has a character that can recognize ferric citrate and taking Fe(III) in. <i>Fec</i> is controlled by <i>Fur</i> and when the density of Fe(III) in the cell is too high, <i>Fur</i> restricts Fec and stops iron uptake.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In the same way, <i>Efe</i>UOB has a character that can recognize ascorbic acid and taking Fe(II) in. <i>Efe</i>UOB is also controlled by <i>Fur</i> and when the density of Fe(II) in the cell is too high, <i>Fur</i> restricts <i>EfeUOB</i> and stops iron uptake.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Using these characters, and by knocking down/out <i>Fur</i> and <i>fie</i>F, <i>Fec</i>/<i>Efe</i>UOB expression wouldn't be restricted so the amount of iron coming in would increase and the amount of iron going out would decrease. The system would work like when iron is lacking.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;As a result, the amount of iron inside <i>E. coli</i> would increase.</p><br />
<br><center><img src="https://static.igem.org/mediawiki/2013/f/f8/Uptake_image.PNG" width="723px"height="305px"></center><br><br />
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<h3 style="background-color:#ffdead ">2.Materials&Methods</h3><br />
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<h3 style="background-color:#f0ffff ">2.1.plasmid construct</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We constructed four plasmids knocking down <i>fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. at the same time, in order to confirm the function of dCas9, we constructed a plasmid knocking down <i>lacZ</i>.<br><br />
プラスミドについて書く<br><br />
<br />
<br />
<br />
<a href="#">Part link</a><br><br><br />
</p><br />
<h3 style="background-color:#f0ffff ">2.2.Evaluation of <i>Fur</i>,<i>fie</i>F knockdown</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We performed two experiments about CRISPRi system in order to confirm the knockdown function as desired. The purpose is a function check for <i>lacZ</i>, <i>Fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. <br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/7/7f/Chiba.CRISPRi.kan.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 1</b> the method of function check about <i>fur</i></p></center><br />
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<a href="https://2013.igem.org/Team:Chiba/Assay/uptake">Assay</a><br />
</p><br />
<h3 style="background-color:#f0ffff ">2.3.Evaluation of absorbed iron volume</h3><br />
<p><br />
<b>Experiment:</b><br><br />
<br />
<br />
<left><img src="https://static.igem.org/mediawiki/2013/3/3f/CRISPRi.PNG" width="409px"height="88px"></left><br />
<right><img src="https://static.igem.org/mediawiki/2013/2/23/DCas9.PNG" width="306px"height="110px"></right><br><br />
<center><b>Fig. a</b></center><br />
<br><p>&nbsp;&nbsp;&nbsp;&nbsp;<i>E. coli</i> stain BL21 was transformed by Plasmid shown in <b>Fig. a</b> .&nbsp;Then we cultured all transformants with atC.&nbsp; atC was added to knock down <i>fur</i> and <i>fie</i>F.&nbsp; After that we cultured it in the presence of ferric citrate, and measured the density of iron that weren’t taken in to <i>E. coli</i> and remained in the medium by measuring Absorbance (Abs 756) with color reagent.</p><br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<p><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid建設の説明</h3><br />
<br />
<h3 style="background-color:#f0ffff ">3.2.Function Check</h3><br />
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<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/26/Chiba.CRISPRi.spot.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 2</b>CRISPRi efficiently silence transcription</p></center><br><br />
<br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/a/a6/Chiba.CRISPRi-lacZ.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 3</b> Function check about CRISPRi-<i>lacZ</i></p></center><br><br />
<br />
1) In the absence of anhydro Tetracycline (aTc), CRISPRi-medeated <i>lac</i>Z gene transcriptional silencing did not occur, resulting in blue-colored colony in which <i>lac</i>Z was expressed.<br><br />
2) In the presence of aTc, CRISPRi-medeated <i>lac</i>Z gene transcriptional silencing successfully occurred, resulting in colorless colony in which <i>lac</i>Z was not expressed.<br><br />
3) CRISPRi-medeated <i>fur</i> gene transcriptional silencing successfully occurred, resulting in the loss of kanamysin resistance.<br><br />
<br />
<br />
<br><br />
<br />
<br />
<br />
<br />
<h3 style="background-color:#f0ffff ">3.3.A knockdown of <i>fie</i>F or fur has no effect on iron uptake </h3><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/f/f0/Chiba_shimamura_special.png"width="800px"></center><br><br />
<center>Fig. 4 Absorbance at each cell number of E. coli(BL21 and SHuffle® introduced each plusmid</center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/5/5e/Chiba_shimamura_kennryousen.png"width="800px"></center><br><br />
<center>Fig. 5 Absorbance of as a function of each iron concentration</center><br><br />
<br />
<br />
Iron uptake was not detectable (iron concentration changes in media were less than micro molar order), even if <i>fur</i> or <i>fie</i>F was successfully knocked down.<br><br />
A future subject is to experiment <br />
今後の課題としては、さらに多い細胞数での定量、または、加える鉄試薬の濃度をさらに調整しなおす必要がある。<br><br />
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<h3 style="background-color:#ffdead ">4.Conclusion</h3><br />
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それは・・<br />
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<h2 id="storage" style="background-color:#ff9933"><center>Sequestration: Fe-storage machine</center></h2><br />
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<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
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<br>&nbsp;&nbsp;&nbsp;&nbsp; <p><b>Fe must be isolated</b>: In order to magnetize <i>E. coli</i>, we need to stuff as much Fe ions as possible in <i>E. coli</i>. Fe(II) could cause Fenton reaction in response to hydrogen peroxide, harmful hydroxyl radicals (OH・). Dilemma is that, feeding too much Fe into cell would kills the host cell. We decided to over express the ferritins that capture and store Fe irons.</p> <br><br />
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<p><b>Fe container machinery</b>: Ferritin particles are made of two small protein subunits (heavy chain (FTH) and light chain (FTL)) (Fig. 1). When expressed, the ferritin subunits automatically assemble into 24-membered protein cages.</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ferrittin.png"alt=""align="middle"></center><br />
<center>Fig. 1. Complex structure of ferritin<br></center><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Heavy chain catalyzes oxidation of iron and stimulate 2Fe(II)+O2→[Fe(III)-O-O-Fe(III)] reaction<br />
<br><br />
<br><br />
2Fe<sub>2</sub>+O<sub>2</sub>+(H<sub>2</sub>O)x+3→Fe<sub>2</sub>O<sub>2</sub>(H<sub>2</sub>O)x+4H+H<sub>2</sub>O<sub>2</sub><br />
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<br><br>&nbsp;&nbsp;&nbsp;&nbsp;On the other hand, light chain takes up Fe(III). By functional expression of these two polypeptides, virtual concentration of Fe inside cell can be much reduced. <br />
Interestingly, the storage capacity, the complex size, and FTH/FTL ratio can vary from species to species. Generally speaking, the mammalian ferritin complex contains more FTL than FTH, while the ferritin complex from bacteria have reversed compositions. <br />
<br><br />
<br><br />
<b>Choice of ferritin</b>: Because the storage capacity of <i>E. coli</i> ferritin is far less than that of mammarian type, we decided to make BioBrick for the functional expression of human ferritin in <i>E. coli</i>.<br />
<br><br />
<br><br />
<b>Hypothesis</b>: By storing Fe in isolation, the maximum capacity for Fe storage should be elevated. Also, the apparent iron tolerance of <i>E. coli</i> should be also elevated. <br />
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<h3 style="background-color:#ffdead ">2.Experiments</h3><br />
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<h3 style="background-color:#f0ffff ">2.1.BioBrick construction</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;The ratio of FTH/FTL can be flexible in ferritin complex, and there exist a best composition that gives the highest Fe-storage activity. In heterologous expression of ferritin, the translational efficiency can be fine-tuned so that we could achieve that best composition. So, we constructed BioBricks for the functional expression of human ferritin complex in two formats; <br />
<br>BBa_K1057002: 'middle' RBS assigned for FTH <br />
<br>BBa_K1057009: 'strong' RBS assigned for FTH <br />
<br>In both construct, two ferritin genes (FTH and FTL) are placed under pBAD promoter to control the timing and expression level of these genes. To facilitate this construction process, we modified an existing Biobrick (BBa_I74608 deposited by iGEM 2007 team Cambridge) into the new BioBrick(BBa_K1057012). This enabled us the rapid, in-parallel, and one-pot digestion/ ligation using Golden gate method. <br />
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<center><img src="https://static.igem.org/mediawiki/2013/3/32/Chiba.ferritin.png"alt=""align="middle"></center><br />
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<center><p>Fig. 2. Cloning procedure of ferritin-producing BioBrick</p></center><br><br />
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<a href="https://2013.igem.org/Team:Chiba/Parts"> Parts link</a><br />
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<h3 style="background-color:#f0ffff ">2.2. Confirmation of Ferritin Expression </h3><br />
<p><br />
Our new BioBricks BBa_K1057002 and BBa_K1057009 were transfected into in BL21 strain. The expression level of each individual components (H-chain and L-chain) was checked by SDS-PAGE/ coomassie blue. As a control, we also conducted the same experiment with “sfgfp generator”.<br />
<br><br />
The results are shown in Fig. 3. Both in total protein fraction (gel in left) and soluble fraction(gel in right), there observed two characteristic bands: one was with the size of 20 kDa (corresonding to FTH), while the other was 19kDa (corresponding to FTL). These bands were not detectable for the control sample (Lane 3) expressing the control gene sfGFP under the control of pBAD promoter.<br />
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<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ike.png" width="750px"height="500px"></center><br><br />
<center><p>Fig. 3. Expression of ferritin in <i>E. coli</i> treated with arabinose. <br />
<br> lane 1. pBAD/araC-ferritin-strong(BBa_K1057009)<br />
<br>lane 2. pBAD/araC-ferritin-mid(BBA_K1057002)<br />
<br>lane 3. BBa_I746908(sfgfp)<br />
</p></center><br />
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</p><br />
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<h3 style="background-color:#f0ffff ">2.3.Evaluation of iron tolerance</h3><br />
<p><br />
<a href="https://2013.igem.org/Team:Chiba/Assay/store">Assay</a><br><br />
Experiment: We constructed two kinds of plasmids with which RBS scores are different as shown in Fig. 2. E. coli strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with above plasmids. Human ferritin genes (fth1 and ftl) are placed on the high-copy plasmid under the control of BAD promoter. To express Human ferritin proteins, arabinose was added into media(final conc. 0.2%). The resultant “ferritin generators” were cultured in the presence of iron citrate (Fe3+) or iron ascorbate (Fe2+), and checked final cell density and colony forming efficiency. As a control, we conducted the same experiment with “sfgfp generator”. <br />
</p><br />
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<h3 style="background-color:#f0ffff ">2.4.Evaluation of magnetism</h3><br />
<p><br />
We examine whether BL21 overexpressing ferritin is attracted to a magnet.<br><br />
Our experimental setup is shown below(Fig. 3).<br><br />
<br />
The details are shown in <a href="https://2013.igem.org/Team:Chiba/Assay/store">assay.</a><br><br />
If <i>E. coli</i> have magnetism, the attract to magnets!<br><br />
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<center><img src="https://static.igem.org/mediawiki/2013/e/e5/Chiba_magnet_method_ssp.png" width="600px"height="400px"></center><br><br />
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<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid</h3><br />
<br />
<h3 style="background-color:#f0ffff ">3.2 Assessment of iron tolerance</h3><br />
<br><p><b>Result. 1</b><br />
<br><left><img src="https://static.igem.org/mediawiki/2013/7/7f/Strorage_BL21_asc_OD_cor.PNG" width="377px"height="289px"></left><br />
<right><img src="https://static.igem.org/mediawiki/2013/f/f5/Storage_SHuffle_asc_OD_cor.PNG" width="372px"height="288px"></right><br><br />
<br><center>Fig. 4</center><br />
<br><br />
<br><p>Fig. 4 shows the optical density of <i>E. coli</i> which is cultured in the presence of iron for 12h (at 37°C).<br><br />
(Approximately 10^7 cells inoculated in to fresh media(2 mL, containing iron)in each.)<br />
<br>・Overexpression of ferritin didn’t affect the growth so much.<br />
<br>・The one without overexpression of ferritin, growth inhibition was observed when the concentration of ferrous ascorbate is 6-8mM. And when the concentration of ferrous ascorbate was over 8 mM, the growth of <i>E. coli</i> wasn’t observed at all.</p><br />
<br>・一方,フェリチンを過剰発現した大腸菌は,アスコルビン酸鉄濃度5-7 mMの範囲では,アスコルビン酸鉄不在下よりも増殖がよく?~ちょっとここ考えます,また9-10 mMのアスコルビン酸鉄濃度においても,細胞は増殖した<br />
<br>・<i>E. coli</i> strain <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> showed same results.<br />
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<br><br><b>Result. 2</b><br />
<br><center><img src="https://static.igem.org/mediawiki/2013/3/39/Storage_result_BL_asc.PNG" width="716px"height="299px"></center><br><br />
<br>The ability to form colony changed similarly.<br />
<br><br />
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<h3 style="background-color:#f0ffff ">3.3 Evaluation of magnetism</h3><br />
<p><b>Result</b><br />
<center><img src="https://static.igem.org/mediawiki/2013/9/93/Chiba_tetsu_BL21_str_ssp.png" width="571px"></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/b/b2/Chiba_tetsu_BL21_mid_sp.png" width="571px"></center><br><br />
<p>Each <i>E. coli</i> don`t move at all.<br><br />
We consider this cause is uptake quantity of Fe(iii) is not sufficiently.<br><br />
So, expression levels of ferritin in each <i>E. coli</i> is not sufficiently.<br><br />
Future subject is to increase uptake quantity of Fe(iii) to knock down/out <i>Fur</i> and <i>fie</i>F.</p><br><br />
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<h2 id="uptake" style="background-color:#ff9933"><center>Uptake</center></h2><br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<br />
<p><br />
<h3 style="background-color:#f0ffff ">1.1.<i>Fur</i></h3><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;<i>Fur</i> (Ferric uptake regulator) controls iron metabolism such as taking iron in or storing iron.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In most cases, it combines with <i>Fur</i> box (which is near the promoter) and regulates transcription.</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;When iron is rich, <i>Fur</i> becomes active and when <i>Fur</i> becomes active, it restricts the expression of iron transporter, and that means that the iron uptake would stop. At the same time,<i> Fur</i> restricts the expression of <i>Ryh</i>B. <i>RyhB</i> is one of the sRNA that restricts the expression of Ferritin, so making <i>Fur</i> active leads ferritin to express.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Conversely, when iron is lacking, <i>Fur</i> becomes inactive, and the iron transporter would work normally, so iron would be taken in. <i>Ryh</i>B would also work normally, so expression of ferritin is restricted and Ferritin wouldn't be expressed.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In short, if we knockdown/knockout <i>Fur</i>, iron transporter would be active so iron would be taken in, but the expression of Ferritin is stopped by <i>Ryh</i>B.</p><br><br />
<br><br />
<h3 style="background-color:#f0ffff ">1.2.<i>fie</i>F</h3><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;There is a Regulator called <i>fie</i>F (ferric iron efflux). It makes iron and zinc flow out of cell and controls detoxification of cell. When <i>fie</i>F is knocked down/out, the tolerance of cell to iron would be lowered.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;<i>Fec</i> has a character that can recognize ferric citrate and taking Fe(III) in. <i>Fec</i> is controlled by <i>Fur</i> and when the density of Fe(III) in the cell is too high, <i>Fur</i> restricts Fec and stops iron uptake.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In the same way, <i>Efe</i>UOB has a character that can recognize ascorbic acid and taking Fe(II) in. <i>Efe</i>UOB is also controlled by <i>Fur</i> and when the density of Fe(II) in the cell is too high, <i>Fur</i> restricts <i>EfeUOB</i> and stops iron uptake.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Using these characters, and by knocking down/out <i>Fur</i> and <i>fie</i>F, <i>Fec</i>/<i>Efe</i>UOB expression wouldn't be restricted so the amount of iron coming in would increase and the amount of iron going out would decrease. The system would work like when iron is lacking.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;As a result, the amount of iron inside <i>E. coli</i> would increase.</p><br />
<br><img src="https://static.igem.org/mediawiki/2013/f/f8/Uptake_image.PNG" width="723px"height="305px"><br><br />
<br />
</p><br />
<br />
<br />
<h3 style="background-color:#ffdead ">2.Materials&Methods</h3><br />
<br />
<h3 style="background-color:#f0ffff ">2.1.plasmid construct</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We constructed four plasmids knocking down <i>fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. at the same time, in order to confirm the function of dCas9, we constructed a plasmid knocking down <i>lacZ</i>.<br><br />
プラスミドについて書く<br><br />
<br />
<br />
<br />
<a href="#">Part link</a><br><br><br />
</p><br />
<h3 style="background-color:#f0ffff ">2.2.Evaluation of <i>Fur</i>,<i>fie</i>F knockdown</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We performed two experiments about CRISPRi system in order to confirm the knockdown function as desired. The purpose is a function check for <i>lacZ</i>, <i>Fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. <br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/7/7f/Chiba.CRISPRi.kan.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 1</b> the method of function check about <i>fur</i></p></center><br />
<br />
<br />
<br />
<a href="https://2013.igem.org/Team:Chiba/Assay/uptake">Assay</a><br />
</p><br />
<h3 style="background-color:#f0ffff ">2.3.Evaluation of absorbed iron volume</h3><br />
<p><br />
<b>Experiment:</b><br><br />
<br />
<br />
<left><img src="https://static.igem.org/mediawiki/2013/3/3f/CRISPRi.PNG" width="409px"height="88px"></left><br />
<right><img src="https://static.igem.org/mediawiki/2013/2/23/DCas9.PNG" width="306px"height="110px"></right><br><br />
<center><b>Fig. a</b></center><br />
<br><p>&nbsp;&nbsp;&nbsp;&nbsp;<i>E. coli</i> stain BL21 was transformed by Plasmid shown in <b>Fig. a</b> .&nbsp;Then we cultured all transformants with atC.&nbsp; atC was added to knock down <i>fur</i> and <i>fie</i>F.&nbsp; After that we cultured it in the presence of ferric citrate, and measured the density of iron that weren’t taken in to <i>E. coli</i> and remained in the medium by measuring Absorbance (Abs 756) with color reagent.</p><br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<p><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid建設の説明</h3><br />
<br />
<h3 style="background-color:#f0ffff ">3.2.Function Check</h3><br />
<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/26/Chiba.CRISPRi.spot.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 2</b>CRISPRi efficiently silence transcription</p></center><br><br />
<br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/a/a6/Chiba.CRISPRi-lacZ.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 3</b> Function check about CRISPRi-<i>lacZ</i></p></center><br><br />
<br />
1) In the absence of anhydro Tetracycline (aTc), CRISPRi-medeated <i>lac</i>Z gene transcriptional silencing did not occur, resulting in blue-colored colony in which <i>lac</i>Z was expressed.<br><br />
2) In the presence of aTc, CRISPRi-medeated <i>lac</i>Z gene transcriptional silencing successfully occurred, resulting in colorless colony in which <i>lac</i>Z was not expressed.<br><br />
3) CRISPRi-medeated <i>fur</i> gene transcriptional silencing successfully occurred, resulting in the loss of kanamysin resistance.<br><br />
<br />
<br />
<br><br />
<br />
<br />
<br />
<br />
<h3 style="background-color:#f0ffff ">3.3.A knockdown of <i>fie</i>F or fur has no effect on iron uptake </h3><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/f/f0/Chiba_shimamura_special.png"width="800px"></center><br><br />
<center>Fig. 4 BL21およびSHuffleでの、各プラスミドを導入した大腸菌の鉄試薬別のabsorbance</center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/5/5e/Chiba_shimamura_kennryousen.png"width="800px"></center><br><br />
<center>Fig. 4 BL21およびSHuffleでの、各プラスミドを導入した大腸菌の鉄試薬別のabsorbance</center><br><br />
<br />
<br />
Iron uptake was not detectable (iron concentration changes in media were less than micro molar order), even if <i>fur</i> or <i>fie</i>F was successfully knocked down.<br><br />
A future subject is to experiment <br />
今後の課題としては、さらに多い細胞数での定量、または、加える鉄試薬の濃度をさらに調整しなおす必要がある。<br><br />
<br />
<br />
<br />
<br />
</p><br />
<h3 style="background-color:#ffdead ">4.Conclusion</h3><br />
<br />
<p><br />
それは・・<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
</div><br />
</body><br />
<br />
</html></div>T.Sendahttp://2013.igem.org/File:Chiba_shimamura_special.pngFile:Chiba shimamura special.png2013-09-28T00:51:52Z<p>T.Senda: </p>
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<div></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/uptakeTeam:Chiba/Project/uptake2013-09-28T00:09:48Z<p>T.Senda: </p>
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<h2 id="uptake" style="background-color:#ff9933"><center>Uptake</center></h2><br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<br />
<p><br />
<h3 style="background-color:#f0ffff ">1.1.<i>Fur</i></h3><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;<i>Fur</i> (Ferric uptake regulator) controls iron metabolism such as taking iron in or storing iron.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In most cases, it combines with <i>Fur</i> box (which is near the promoter) and regulates transcription.</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;When iron is rich, <i>Fur</i> becomes active and when <i>Fur</i> becomes active, it restricts the expression of iron transporter, and that means that the iron uptake would stop. At the same time,<i> Fur</i> restricts the expression of <i>Ryh</i>B. <i>RyhB</i> is one of the sRNA that restricts the expression of Ferritin, so making <i>Fur</i> active leads ferritin to express.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Conversely, when iron is lacking, <i>Fur</i> becomes inactive, and the iron transporter would work normally, so iron would be taken in. <i>Ryh</i>B would also work normally, so expression of ferritin is restricted and Ferritin wouldn't be expressed.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In short, if we knockdown/knockout <i>Fur</i>, iron transporter would be active so iron would be taken in, but the expression of Ferritin is stopped by <i>Ryh</i>B.</p><br><br />
<br><br />
<h3 style="background-color:#f0ffff ">1.2.<i>fie</i>F</h3><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;There is a Regulator called <i>fie</i>F (ferric iron efflux). It makes iron and zinc flow out of cell and controls detoxification of cell. When <i>fie</i>F is knocked down/out, the tolerance of cell to iron would be lowered.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;<i>Fec</i> has a character that can recognize ferric citrate and taking Fe(III) in. <i>Fec</i> is controlled by <i>Fur</i> and when the density of Fe(III) in the cell is too high, <i>Fur</i> restricts Fec and stops iron uptake.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In the same way, <i>Efe</i>UOB has a character that can recognize ascorbic acid and taking Fe(II) in. <i>Efe</i>UOB is also controlled by <i>Fur</i> and when the density of Fe(II) in the cell is too high, <i>Fur</i> restricts <i>EfeUOB</i> and stops iron uptake.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Using these characters, and by knocking down/out <i>Fur</i> and <i>fie</i>F, <i>Fec</i>/<i>Efe</i>UOB expression wouldn't be restricted so the amount of iron coming in would increase and the amount of iron going out would decrease. The system would work like when iron is lacking.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;As a result, the amount of iron inside <i>E. coli</i> would increase.</p><br />
<br />
</p><br />
<br />
<br />
<h3 style="background-color:#ffdead ">2.Materials&Methods</h3><br />
<br />
<h3 style="background-color:#f0ffff ">2.1.plasmid construct</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We constructed four plasmids knocking down <i>fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. at the same time, in order to confirm the function of dCas9, we constructed a plasmid knocking down <i>lacZ</i>.<br><br />
プラスミドについて書く<br><br />
<br />
<br />
<br />
<a href="#">Part link</a><br><br><br />
</p><br />
<h3 style="background-color:#f0ffff ">2.2.Evaluation of <i>Fur</i>,<i>fie</i>F knockdown</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We performed two experiments about CRISPRi system in order to confirm the knockdown function as desired. The purpose is a function check for <i>lacZ</i>, <i>Fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. <br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/7/7f/Chiba.CRISPRi.kan.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 1</b> the method of function check about <i>fur</i></p></center><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/2/26/Chiba.CRISPRi.spot.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 2</b><i>CRISPR</i>i efficiently silence transcription</p></center><br><br />
<br />
<br />
<br><img src="https://static.igem.org/mediawiki/2013/f/f8/Uptake_image.PNG" width="723px"height="305px"><br><br><br />
<br />
<a href="https://2013.igem.org/Team:Chiba/Assay/uptake">Assay</a><br />
</p><br />
<h3 style="background-color:#f0ffff ">2.3.Evaluation of absorbed iron volume</h3><br />
<p><br />
<b>Experiment:</b><br><br />
<br />
<br />
<left><img src="https://static.igem.org/mediawiki/2013/3/3f/CRISPRi.PNG" width="409px"height="88px"></left><br />
<right><img src="https://static.igem.org/mediawiki/2013/2/23/DCas9.PNG" width="306px"height="110px"></right><br><br />
<center><b>Fig. 2</b></center><br />
<br><p>&nbsp;&nbsp;&nbsp;&nbsp;E. coli stain BL21 was transformed by Plasmid shown in <b>Fig. 2</b> .&nbsp;Then we cultured all transformants with atC.&nbsp; atC was added to knock down <i>fur</i> and <i>fie</i>F.&nbsp; After that we cultured it in the presence of ferric citrate, and measured the density of iron that weren’t taken in to <i>E. coli</i> and remained in the medium by measuring Absorbance (Abs 756) with color reagent(Fig. 2).</p><br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<p><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid建設の説明</h3><br />
<br />
<h3 style="background-color:#f0ffff ">3.2.Function Check</h3><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/6/63/Chiba.CRISPRi.lacZ.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 3</b> Function check about CRISPRi-<i>lacZ</i></p></center><br><br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp;aTcがないときCRISPRi-lacZは発現されないためlacZの抑制は起こらず,X-galは分解されて培地は青く染まった。一方,aTcがあるとCRISPRi-lacZによる抑制が起こるため,lacZは発現されず,培地は白いままとなった。このことから,CRISPRi-lacZの機能を確認することができた。<br><br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp;一方,fur, fieF, trxB, およびgor遺伝子を狙ったgRNAについて,その標的配列の下流にカナマイシン遺伝子を挿入した大腸菌株の作製を試みた。すると,furおよびtrxBの下流にカナマイシン遺伝子を挿入した株(それぞれMG1655Δfur::kmrおよびMG1655ΔtrxB::kmr)が得られた。一方,fieFおよびgorの下流にカナマイシン遺伝子を挿入した株(それぞれMG1655ΔfieF::kmrおよびMG1655Δgor::kmr)は得られなかった。これは,trxBおよびgorのプロモータが弱く,カナマイシン耐性遺伝子の発現量が,大腸菌にカナマイシン耐性を与えるほど多くないためと考えられる。<br><br />
<br />
<br />
<br />
<br />
<h3 style="background-color:#f0ffff ">3.3.鉄取り込み量の評価</h3><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/f/f8/Chiba_shimamurasan.png"width="800px"></center><br><br />
<center>Fig. 4 BL21およびSHuffleでの、各プラスミドを導入した大腸菌の鉄試薬別のabsorbance</center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/5/5e/Chiba_shimamura_kennryousen.png"width="800px"></center><br><br />
<center>Fig. 4 BL21およびSHuffleでの、各プラスミドを導入した大腸菌の鉄試薬別のabsorbance</center><br><br />
<br />
<br />
<br />
<br />
BL21、Shuffleともに、各細胞数において、鉄溶液の種類にかかわらず、吸光度にばらつきがでた。<br><br />
これより、この実験方法では、大腸菌の取り込んだ鉄量が少なすぎるため、定量できなかったと考えられる。<br><br />
今後の課題としては、さらに多い細胞数での定量、または、加える鉄試薬の濃度をさらに調整しなおす必要がある。<br><br />
また、細胞破砕によって大腸内の鉄量を定量する方法も検討する必要がある。<br><br />
<br />
<br />
<br />
</p><br />
<h3 style="background-color:#ffdead ">4.Conclusion</h3><br />
<br />
<p><br />
それは・・<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
</div><br />
</body><br />
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</html></div>T.Sendahttp://2013.igem.org/File:Chiba_shimamura_kennryousen.pngFile:Chiba shimamura kennryousen.png2013-09-28T00:04:04Z<p>T.Senda: </p>
<hr />
<div></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/uptakeTeam:Chiba/Project/uptake2013-09-27T23:54:32Z<p>T.Senda: </p>
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<head><br />
<meta http-equiv="Content-Type" content="text/html;charset=utf-8"/><br />
<title>iGEM-2013 Chiba</title><br />
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<style type="text/css"><br />
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<h2 id="uptake" style="background-color:#ff9933"><center>Uptake</center></h2><br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<br />
<p><br />
<h3 style="background-color:#f0ffff ">1.1.<i>Fur</i></h3><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;<i>Fur</i> (Ferric uptake regulator) controls iron metabolism such as taking iron in or storing iron.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In most cases, it combines with <i>Fur</i> box (which is near the promoter) and regulates transcription.</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;When iron is rich, <i>Fur</i> becomes active and when <i>Fur</i> becomes active, it restricts the expression of iron transporter, and that means that the iron uptake would stop. At the same time,<i> Fur</i> restricts the expression of <i>Ryh</i>B. <i>RyhB</i> is one of the sRNA that restricts the expression of Ferritin, so making <i>Fur</i> active leads ferritin to express.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Conversely, when iron is lacking, <i>Fur</i> becomes inactive, and the iron transporter would work normally, so iron would be taken in. <i>Ryh</i>B would also work normally, so expression of ferritin is restricted and Ferritin wouldn't be expressed.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In short, if we knockdown/knockout <i>Fur</i>, iron transporter would be active so iron would be taken in, but the expression of Ferritin is stopped by <i>Ryh</i>B.</p><br><br />
<br><br />
<h3 style="background-color:#f0ffff ">1.2.<i>fie</i>F</h3><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;There is a Regulator called <i>fie</i>F (ferric iron efflux). It makes iron and zinc flow out of cell and controls detoxification of cell. When <i>fie</i>F is knocked down/out, the tolerance of cell to iron would be lowered.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;<i>Fec</i> has a character that can recognize ferric citrate and taking Fe(III) in. <i>Fec</i> is controlled by <i>Fur</i> and when the density of Fe(III) in the cell is too high, <i>Fur</i> restricts Fec and stops iron uptake.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In the same way, <i>Efe</i>UOB has a character that can recognize ascorbic acid and taking Fe(II) in. <i>Efe</i>UOB is also controlled by <i>Fur</i> and when the density of Fe(II) in the cell is too high, <i>Fur</i> restricts <i>EfeUOB</i> and stops iron uptake.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Using these characters, and by knocking down/out <i>Fur</i> and <i>fie</i>F, <i>Fec</i>/<i>Efe</i>UOB expression wouldn't be restricted so the amount of iron coming in would increase and the amount of iron going out would decrease. The system would work like when iron is lacking.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;As a result, the amount of iron inside <i>E. coli</i> would increase.</p><br />
<br />
</p><br />
<br />
<br />
<h3 style="background-color:#ffdead ">2.Materials&Methods</h3><br />
<br />
<h3 style="background-color:#f0ffff ">2.1.plasmid construct</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We constructed four plasmids knocking down <i>fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. at the same time, in order to confirm the function of dCas9, we constructed a plasmid knocking down <i>lacZ</i>.<br><br />
プラスミドについて書く<br><br />
<br />
<br />
<br />
<a href="#">Part link</a><br><br><br />
</p><br />
<h3 style="background-color:#f0ffff ">2.2.Evaluation of <i>Fur</i>,<i>fie</i>F knockdown</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We performed two experiments about CRISPRi system in order to confirm the knockdown function as desired. The purpose is a function check for <i>lacZ</i>, <i>Fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. <br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/7/7f/Chiba.CRISPRi.kan.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 1</b> the method of function check about <i>fur</i></p></center><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/2/26/Chiba.CRISPRi.spot.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 2</b><i>CRISPR</i>i efficiently silence transcription</p></center><br><br />
<br />
<br />
<br><img src="https://static.igem.org/mediawiki/2013/f/f8/Uptake_image.PNG" width="723px"height="305px"><br><br><br />
<br />
<a href="https://2013.igem.org/Team:Chiba/Assay/uptake">Assay</a><br />
</p><br />
<h3 style="background-color:#f0ffff ">2.3.Evaluation of absorbed iron volume</h3><br />
<p><br />
<b>Experiment:</b><br><br />
<br />
<br />
<left><img src="https://static.igem.org/mediawiki/2013/3/3f/CRISPRi.PNG" width="409px"height="88px"></left><br />
<right><img src="https://static.igem.org/mediawiki/2013/2/23/DCas9.PNG" width="306px"height="110px"></right><br><br />
<center><b>Fig. 2</b></center><br />
<br><p>&nbsp;&nbsp;&nbsp;&nbsp;E. coli stain BL21 was transformed by Plasmid shown in <b>Fig. 2</b> .&nbsp;Then we cultured all transformants with atC.&nbsp; atC was added to knock down <i>fur</i> and <i>fie</i>F.&nbsp; After that we cultured it in the presence of ferric citrate, and measured the density of iron that weren’t taken in to <i>E. coli</i> and remained in the medium by measuring Absorbance (Abs 756) with color reagent(Fig. 2).</p><br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<p><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid建設の説明</h3><br />
<br />
<h3 style="background-color:#f0ffff ">3.2.Function Check</h3><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/6/63/Chiba.CRISPRi.lacZ.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 3</b> Function check about CRISPRi-<i>lacZ</i></p></center><br><br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp;aTcがないときCRISPRi-lacZは発現されないためlacZの抑制は起こらず,X-galは分解されて培地は青く染まった。一方,aTcがあるとCRISPRi-lacZによる抑制が起こるため,lacZは発現されず,培地は白いままとなった。このことから,CRISPRi-lacZの機能を確認することができた。<br><br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp;一方,fur, fieF, trxB, およびgor遺伝子を狙ったgRNAについて,その標的配列の下流にカナマイシン遺伝子を挿入した大腸菌株の作製を試みた。すると,furおよびtrxBの下流にカナマイシン遺伝子を挿入した株(それぞれMG1655Δfur::kmrおよびMG1655ΔtrxB::kmr)が得られた。一方,fieFおよびgorの下流にカナマイシン遺伝子を挿入した株(それぞれMG1655ΔfieF::kmrおよびMG1655Δgor::kmr)は得られなかった。これは,trxBおよびgorのプロモータが弱く,カナマイシン耐性遺伝子の発現量が,大腸菌にカナマイシン耐性を与えるほど多くないためと考えられる。<br><br />
<br />
<br />
<br />
<br />
<h3 style="background-color:#f0ffff ">3.3.鉄取り込み量の評価</h3><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/f/f8/Chiba_shimamurasan.png"width="800px"></center><br><br />
<center>Fig. 4 BL21およびSHuffleでの、各プラスミドを導入した大腸菌の鉄試薬別のabsorbance</center><br><br />
<br />
<br />
BL21、Shuffleともに、各細胞数において、鉄溶液の種類にかかわらず、吸光度にばらつきがでた。<br><br />
これより、この実験方法では、大腸菌の取り込んだ鉄量が少なすぎるため、定量できなかったと考えられる。<br><br />
今後の課題としては、さらに多い細胞数での定量、または、加える鉄試薬の濃度をさらに調整しなおす必要がある。<br><br />
また、細胞破砕によって大腸内の鉄量を定量する方法も検討する必要がある。<br><br />
<br />
<br />
<br />
</p><br />
<h3 style="background-color:#ffdead ">4.Conclusion</h3><br />
<br />
<p><br />
それは・・<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
</div><br />
</body><br />
<br />
</html></div>T.Sendahttp://2013.igem.org/File:Chiba_shimamurasan.pngFile:Chiba shimamurasan.png2013-09-27T23:50:07Z<p>T.Senda: </p>
<hr />
<div></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/storeTeam:Chiba/Project/store2013-09-27T23:41:46Z<p>T.Senda: </p>
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<title>iGEM-2013 Chiba</title><br />
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<h2 id="storage" style="background-color:#ff9933"><center>Sequestration: Fe-storage machine</center></h2><br />
<br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp; <p><b>Fe must be isolated</b>: In order to magnetize <i>E. coli</i>, we need to stuff as much Fe ions as possible in <i>E. coli</i>. Fe(II) could cause Fenton reaction in response to hydrogen peroxide, harmful hydroxyl radicals (OH・). Dilemma is that, feeding too much Fe into cell would kills the host cell. We decided to over express the ferritins that capture and store Fe irons.</p> <br><br />
<br><br />
<p><b>Fe container machinery</b>: Ferritin particles are made of two small protein subunits (heavy chain (FTH) and light chain (FTL)) (Fig. 1). When expressed, the ferritin subunits automatically assemble into 24-membered protein cages.</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ferrittin.png"alt=""align="middle"></center><br />
<center>Fig. 1. Complex structure of ferritin<br></center><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Heavy chain catalyzes oxidation of iron and stimulate 2Fe(II)+O2→[Fe(III)-O-O-Fe(III)] reaction<br />
<br><br />
<br><br />
2Fe<sub>2</sub>+O<sub>2</sub>+(H<sub>2</sub>O)x+3→Fe<sub>2</sub>O<sub>2</sub>(H<sub>2</sub>O)x+4H+H<sub>2</sub>O<sub>2</sub><br />
<br />
<br><br>&nbsp;&nbsp;&nbsp;&nbsp;On the other hand, light chain takes up Fe(III). By functional expression of these two polypeptides, virtual concentration of Fe inside cell can be much reduced. <br />
Interestingly, the storage capacity, the complex size, and FTH/FTL ratio can vary from species to species. Generally speaking, the mammalian ferritin complex contains more FTL than FTH, while the ferritin complex from bacteria have reversed compositions. <br />
<br><br />
<br><br />
<b>Choice of ferritin</b>: Because the storage capacity of <i>E. coli</i> ferritin is far less than that of mammarian type, we decided to make BioBrick for the functional expression of human ferritin in <i>E. coli</i>.<br />
<br><br />
<br><br />
<b>Hypothesis</b>: By storing Fe in isolation, the maximum capacity for Fe storage should be elevated. Also, the apparent iron tolerance of <i>E. coli</i> should be also elevated. <br />
<br />
</br></br><br />
<br><br />
<p><br />
<br />
<br />
<h3 style="background-color:#ffdead ">2.Experiments</h3><br />
<br />
<p><br />
<h3 style="background-color:#f0ffff ">2.1.BioBrick construction</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;The ratio of FTH/FTL can be flexible in ferritin complex, and there exist a best composition that gives the highest Fe-storage activity. In heterologous expression of ferritin, the translational efficiency can be fine-tuned so that we could achieve that best composition. So, we constructed BioBricks for the functional expression of human ferritin complex in two formats; <br />
<br>BBa_K1057002: 'middle' RBS assigned for FTH <br />
<br>BBa_K1057009: 'strong' RBS assigned for FTH <br />
<br>In both construct, two ferritin genes (FTH and FTL) are placed under pBAD promoter to control the timing and expression level of these genes. To facilitate this construction process, we modified an existing Biobrick (BBa_I74608 deposited by iGEM 2007 team Cambridge) into the new BioBrick(BBa_K1057012). This enabled us the rapid, in-parallel, and one-pot digestion/ ligation using Golden gate method. <br />
<br><br />
<br><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/3/32/Chiba.ferritin.png"alt=""align="middle"></center><br />
<br />
<br />
<center><p>Fig. 2. Cloning procedure of ferritin-producing BioBrick</p></center><br><br />
<br><br />
</p><br />
<br />
<br />
<a href="https://2013.igem.org/Team:Chiba/Parts"> Parts link</a><br />
</p><br />
<br />
<br />
<h3 style="background-color:#f0ffff ">2.2.Expression of Ferritin in protein level</h3><br />
<p><br />
Our new BioBricks BBa_K1057002 and BBa_K1057009 were transfected into in BL21 strain. The expression level of each individual components (H-chain and L-chain) was checked by SDS-PAGE/ coomassie blue. As a control, we also conducted the same experiment with “sfgfp generator”.<br />
<br><br />
The results are shown in Fig. 3. Both in total protein fraction (gel in left) and soluble fraction(gel in right), there observed two characteristic bands: one was with the size of 20 kDa (corresonding to FTH), while the other was 19kDa (corresponding to FTL). These bands were not detectable for the control sample (Lane 3) expressing the control gene sfGFP under the control of pBAD promoter.<br />
<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ike.png" width="750px"height="500px"></center><br><br />
<center><p>Fig. 3. Expression of ferritin in <i>E. coli</i> treated with arabinose. <br />
<br> lane 1. pBAD/araC-ferritin-strong(BBa_K1057009)<br />
<br>lane 2. pBAD/araC-ferritin-mid(BBA_K1057002)<br />
<br>lane 3. BBa_I746908(sfgfp)<br />
</p></center><br />
<br />
<br />
<br />
</p><br />
<br />
<h3 style="background-color:#f0ffff ">2.3.Evaluation of iron tolerance</h3><br />
<p><br />
<a href="https://2013.igem.org/Team:Chiba/Assay/store">Assay</a><br><br />
Experiment: We constructed two kinds of plasmids with which RBS scores are different as shown in Fig. 2. E. coli strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with above plasmids. Human ferritin genes (fth1 and ftl) are placed on the high-copy plasmid under the control of BAD promoter. To express Human ferritin proteins, arabinose was added into media(final conc. 0.2%). The resultant “ferritin generators” were cultured in the presence of iron citrate (Fe3+) or iron ascorbate (Fe2+), and checked final cell density and colony forming efficiency. As a control, we conducted the same experiment with “sfgfp generator”. <br />
</p><br />
<br />
<h3 style="background-color:#f0ffff ">2.4.Evaluation of magnetism</h3><br />
<p><br />
We examine whether BL21 overexpressing ferritin is attracted to a magnet.<br><br />
Our experimental setup is shown below(Fig. 3).<br><br />
<br />
The details are shown in <a href="https://2013.igem.org/Team:Chiba/Assay/store">assay.</a><br><br />
If <i>E. coli</i> have magnetism, the attract to magnets!<br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/e/e5/Chiba_magnet_method_ssp.png" width="600px"height="400px"></center><br><br />
<br><br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid</h3><br />
<br />
<h3 style="background-color:#f0ffff ">3.2 Assessment of iron tolerance</h3><br />
<br><p><b>Result. 1</b><br />
<br><left><img src="https://static.igem.org/mediawiki/2013/7/7f/Strorage_BL21_asc_OD_cor.PNG" width="377px"height="289px"></left><br />
<right><img src="https://static.igem.org/mediawiki/2013/f/f5/Storage_SHuffle_asc_OD_cor.PNG" width="372px"height="288px"></right><br><br />
<br><center>Fig. 4</center><br />
<br><br />
<br><p>Fig. 4 shows the optical density of <i>E. coli</i> which is cultured in the presence of iron for 12h (at 37°C).<br><br />
(Approximately 10^7 cells inoculated in to fresh media(2 mL, containing iron)in each.)<br />
<br>・Overexpression of ferritin didn’t affect the growth so much.<br />
<br>・The one without overexpression of ferritin, growth inhibition was observed when the concentration of ferrous ascorbate is 6-8mM. And when the concentration of ferrous ascorbate was over 8 mM, the growth of <i>E. coli</i> wasn’t observed at all.</p><br />
<br>・一方,フェリチンを過剰発現した大腸菌は,アスコルビン酸鉄濃度5-7 mMの範囲では,アスコルビン酸鉄不在下よりも増殖がよく?~ちょっとここ考えます,また9-10 mMのアスコルビン酸鉄濃度においても,細胞は増殖した<br />
<br>・<i>E. coli</i> strain <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> showed same results.<br />
<br />
<br />
<br />
<br><br><b>Result. 2</b><br />
<br><center><img src="https://static.igem.org/mediawiki/2013/3/39/Storage_result_BL_asc.PNG" width="716px"height="299px"></center><br><br />
<br>The ability to form colony changed similarly.<br />
<br><br />
<br />
<h3 style="background-color:#f0ffff ">3.3 Evaluation of magnetism</h3><br />
<p><b>Result</b><br />
<center><img src="https://static.igem.org/mediawiki/2013/9/93/Chiba_tetsu_BL21_str_ssp.png" width="571px"></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/b/b2/Chiba_tetsu_BL21_mid_sp.png" width="571px"></center><br><br />
<p>Each <i>E. coli</i> don`t move at all.<br><br />
We consider this cause is uptake quantity of Fe(iii) is not sufficiently.<br><br />
So, expression levels of ferritin in each <i>E. coli</i> is not sufficiently.<br><br />
Future subject is to increase uptake quantity of Fe(iii) to knock down/out <i>Fur</i> and <i>fie</i>F.</p><br><br />
<br />
<br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">4.Conclusion</h3><br />
<br />
<p><br />
それは・・<br />
<br />
</p><br />
<br />
<br />
<br />
</div><br />
</body><br />
<br />
</html></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/storeTeam:Chiba/Project/store2013-09-27T23:35:04Z<p>T.Senda: </p>
<hr />
<div>{{Chiba_base}}<br />
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<head><br />
<meta http-equiv="Content-Type" content="text/html;charset=utf-8"/><br />
<title>iGEM-2013 Chiba</title><br />
<br />
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<h2 id="storage" style="background-color:#ff9933"><center>Sequestration: Fe-storage machine</center></h2><br />
<br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp; <p><b>Fe must be isolated</b>: In order to magnetize <i>E. coli</i>, we need to stuff as much Fe ions as possible in <i>E. coli</i>. Fe(II) could cause Fenton reaction in response to hydrogen peroxide, harmful hydroxyl radicals (OH・). Dilemma is that, feeding too much Fe into cell would kills the host cell. We decided to over express the ferritins that capture and store Fe irons.</p> <br><br />
<br><br />
<p><b>Fe container machinery</b>: Ferritin particles are made of two small protein subunits (heavy chain (FTH) and light chain (FTL)) (Fig. 1). When expressed, the ferritin subunits automatically assemble into 24-membered protein cages.</p><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ferrittin.png"alt=""align="middle"></center><br />
<center>Fig. 1. Complex structure of ferritin<br></center><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Heavy chain catalyzes oxidation of iron and stimulate 2Fe(II)+O2→[Fe(III)-O-O-Fe(III)] reaction<br />
<br><br />
<br><br />
2Fe<sub>2</sub>+O<sub>2</sub>+(H<sub>2</sub>O)x+3→Fe<sub>2</sub>O<sub>2</sub>(H<sub>2</sub>O)x+4H+H<sub>2</sub>O<sub>2</sub><br />
<br />
<br><br>&nbsp;&nbsp;&nbsp;&nbsp;On the other hand, light chain takes up Fe(III). By functional expression of these two polypeptides, virtual concentration of Fe inside cell can be much reduced. <br />
Interestingly, the storage capacity, the complex size, and FTH/FTL ratio can vary from species to species. Generally speaking, the mammalian ferritin complex contains more FTL than FTH, while the ferritin complex from bacteria have reversed compositions. <br />
<br><br />
<br><br />
<b>Choice of ferritin</b>: Because the storage capacity of <i>E. coli</i> ferritin is far less than that of mammarian type, we decided to make BioBrick for the functional expression of human ferritin in <i>E. coli</i>.<br />
<br><br />
<br><br />
<b>Hypothesis</b>: By storing Fe in isolation, the maximum capacity for Fe storage should be elevated. Also, the apparent iron tolerance of <i>E. coli</i> should be also elevated. <br />
<br />
</br></br><br />
<br><br />
<p><br />
<br />
<br />
<h3 style="background-color:#ffdead ">2.Experiments</h3><br />
<br />
<p><br />
<h3 style="background-color:#f0ffff ">2.1.BioBrick construction</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;The ratio of FTH/FTL can be flexible in ferritin complex, and there exist a best composition that gives the highest Fe-storage activity. In heterologous expression of ferritin, the translational efficiency can be fine-tuned so that we could achieve that best composition. So, we constructed BioBricks for the functional expression of human ferritin complex in two formats; <br />
<br>BBa_K1057002: 'middle' RBS assigned for FTH <br />
<br>BBa_K1057009: 'strong' RBS assigned for FTH <br />
<br>In both construct, two ferritin genes (FTH and FTL) are placed under pBAD promoter to control the timing and expression level of these genes. To facilitate this construction process, we modified an existing Biobrick (BBa_I74608 deposited by iGEM 2007 team Cambridge) into the new BioBrick(BBa_K1057012). This enabled us the rapid, in-parallel, and one-pot digestion/ ligation using Golden gate method. <br />
<br><br />
<br><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/3/32/Chiba.ferritin.png"alt=""align="middle"></center><br />
<br />
<br />
<center><p>Fig. 2. Cloning procedure of ferritin-producing BioBrick</p></center><br><br />
<br><br />
</p><br />
<br />
<br />
<a href="https://2013.igem.org/Team:Chiba/Parts"> Parts link</a><br />
</p><br />
<br />
<br />
<h3 style="background-color:#f0ffff ">2.2.Expression of Ferritin in protein level</h3><br />
<p><br />
Our new BioBricks BBa_K1057002 and BBa_K1057009 were transfected into in BL21 strain. The expression level of each individual components (H-chain and L-chain) was checked by SDS-PAGE/ coomassie blue. As a control, we also conducted the same experiment with “sfgfp generator”.<br />
<br><br />
The results are shown in Fig. 3. Both in total protein fraction (gel in left) and soluble fraction(gel in right), there observed two characteristic bands: one was with the size of 20 kDa (corresonding to FTH), while the other was 19kDa (corresponding to FTL). These bands were not detectable for the control sample (Lane 3) expressing the control gene sfGFP under the control of pBAD promoter.<br />
<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ike.png" width="750px"height="500px"></center><br><br />
<center><p>Fig. 3. Expression of ferritin in <i>E. coli</i> treated with arabinose. <br />
<br> lane 1. pBAD/araC-ferritin-strong(BBa_K1057009)<br />
<br>lane 2. pBAD/araC-ferritin-mid(BBA_K1057002)<br />
<br>lane 3. BBa_I746908(sfgfp)<br />
</p></center><br />
<br />
<br />
<br />
</p><br />
<br />
<h3 style="background-color:#f0ffff ">2.3.Evaluation of iron tolerance</h3><br />
<p><br />
<a href="https://2013.igem.org/Team:Chiba/Assay/store">Assay</a><br><br />
Experiment: We constructed two kinds of plasmids with which RBS scores are different as shown in Fig. 2. E. coli strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with above plasmids. Human ferritin genes (fth1 and ftl) are placed on the high-copy plasmid under the control of BAD promoter. To express Human ferritin proteins, arabinose was added into media(final conc. 0.2%). The resultant “ferritin generators” were cultured in the presence of iron citrate (Fe3+) or iron ascorbate (Fe2+), and checked final cell density and colony forming efficiency. As a control, we conducted the same experiment with “sfgfp generator”. <br />
</p><br />
<br />
<h3 style="background-color:#f0ffff ">2.4.Evaluation of magnetism</h3><br />
<p><br />
We examine whether BL21 overexpressing ferritin is attracted to a magnet.<br><br />
Our experimental setup is shown below(Fig. 3).<br><br />
<br />
The details are shown in <a href="https://2013.igem.org/Team:Chiba/Assay/store">assay.</a><br><br />
If <i>E. coli</i> have magnetism, the attract to magnets!<br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/e/e5/Chiba_magnet_method_ssp.png" width="600px"height="400px"></center><br><br />
<br><br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid</h3><br />
<br />
<h3 style="background-color:#f0ffff ">3.2 Assessment of iron tolerance</h3><br />
<br><p><b>Result. 1</b><br />
<br><left><img src="https://static.igem.org/mediawiki/2013/7/7f/Strorage_BL21_asc_OD_cor.PNG" width="377px"height="289px"></left><br />
<right><img src="https://static.igem.org/mediawiki/2013/f/f5/Storage_SHuffle_asc_OD_cor.PNG" width="372px"height="288px"></right><br><br />
<br><center>Fig. 4</center><br />
<br><br />
<br><p>Fig. 4 shows the optical density of <i>E. coli</i> which is cultured in the presence of iron for 12h (at 37°C).<br><br />
(Approximately 10^7 cells inoculated in to fresh media(2 mL, containing iron)in each.)<br />
<br>・Overexpression of ferritin didn’t affect the growth so much.<br />
<br>・The one without overexpression of ferritin, growth inhibition was observed when the concentration of ferrous ascorbate is 6-8mM. And when the concentration of ferrous ascorbate was over 8 mM, the growth of <i>E. coli</i> wasn’t observed at all.</p><br />
<br>・一方,フェリチンを過剰発現した大腸菌は,アスコルビン酸鉄濃度5-7 mMの範囲では,アスコルビン酸鉄不在下よりも増殖がよく?~ちょっとここ考えます,また9-10 mMのアスコルビン酸鉄濃度においても,細胞は増殖した<br />
<br>・<i>E. coli</i> strain <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> showed same results.<br />
<br />
<br />
<br />
<br><br><b>Result. 2</b><br />
<br><center><img src="https://static.igem.org/mediawiki/2013/3/39/Storage_result_BL_asc.PNG" width="716px"height="299px"></center><br><br />
<br>The ability to form colony changed similarly.<br />
<br><br />
<br />
<h3 style="background-color:#f0ffff ">3.3 Evaluation of magnetism</h3><br />
<p><b>Result</b><br />
<center><img src="https://2013.igem.org/File:Chiba_tetsu_BL21_str_ssp.png" width="571px"height="500px"></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/b/b2/Chiba_tetsu_BL21_mid_sp.png" width="571px"height="500px"></center><br><br />
<p>Each <i>E. coli</i> don`t move at all.<br><br />
We consider this cause is uptake quantity of Fe(iii) is not sufficiently.<br><br />
So, expression levels of ferritin in each <i>E. coli</i> is not sufficiently.<br><br />
Future subject is to increase uptake quantity of Fe(iii) to knock down/out <i>Fur</i> and <i>fie</i>F.</p><br><br />
<br />
<br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">4.Conclusion</h3><br />
<br />
<p><br />
それは・・<br />
<br />
</p><br />
<br />
<br />
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</html></div>T.Sendahttp://2013.igem.org/File:Chiba_tetsu_BL21_mid_sp.pngFile:Chiba tetsu BL21 mid sp.png2013-09-27T23:32:45Z<p>T.Senda: </p>
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<div></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/uptakeTeam:Chiba/Project/uptake2013-09-27T23:13:35Z<p>T.Senda: </p>
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<h2 id="uptake" style="background-color:#ff9933"><center>Uptake</center></h2><br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
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<p><br />
<h3 style="background-color:#f0ffff ">1.1.<i>Fur</i></h3><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;<i>Fur</i> (Ferric uptake regulator) controls iron metabolism such as taking iron in or storing iron.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In most cases, it combines with <i>Fur</i> box (which is near the promoter) and regulates transcription.</p><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;When iron is rich, <i>Fur</i> becomes active and when <i>Fur</i> becomes active, it restricts the expression of iron transporter, and that means that the iron uptake would stop. At the same time,<i> Fur</i> restricts the expression of <i>Ryh</i>B. <i>RyhB</i> is one of the sRNA that restricts the expression of Ferritin, so making <i>Fur</i> active leads ferritin to express.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Conversely, when iron is lacking, <i>Fur</i> becomes inactive, and the iron transporter would work normally, so iron would be taken in. <i>Ryh</i>B would also work normally, so expression of ferritin is restricted and Ferritin wouldn't be expressed.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In short, if we knockdown/knockout <i>Fur</i>, iron transporter would be active so iron would be taken in, but the expression of Ferritin is stopped by <i>Ryh</i>B.</p><br><br />
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<h3 style="background-color:#f0ffff ">1.2.<i>fie</i>F</h3><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;There is a Regulator called <i>fie</i>F (ferric iron efflux). It makes iron and zinc flow out of cell and controls detoxification of cell. When <i>fie</i>F is knocked down/out, the tolerance of cell to iron would be lowered.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;<i>Fec</i> has a character that can recognize ferric citrate and taking Fe(III) in. <i>Fec</i> is controlled by <i>Fur</i> and when the density of Fe(III) in the cell is too high, <i>Fur</i> restricts Fec and stops iron uptake.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In the same way, <i>Efe</i>UOB has a character that can recognize ascorbic acid and taking Fe(II) in. <i>Efe</i>UOB is also controlled by <i>Fur</i> and when the density of Fe(II) in the cell is too high, <i>Fur</i> restricts <i>EfeUOB</i> and stops iron uptake.<br><br />
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&nbsp;&nbsp;&nbsp;&nbsp;Using these characters, and by knocking down/out <i>Fur</i> and <i>fie</i>F, <i>Fec</i>/<i>Efe</i>UOB expression wouldn't be restricted so the amount of iron coming in would increase and the amount of iron going out would decrease. The system would work like when iron is lacking.<br><br />
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&nbsp;&nbsp;&nbsp;&nbsp;As a result, the amount of iron inside <i>E. coli</i> would increase.</p><br />
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<h3 style="background-color:#ffdead ">2.Materials&Methods</h3><br />
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<h3 style="background-color:#f0ffff ">2.1.plasmid construct</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We constructed four plasmids knocking down <i>fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. at the same time, in order to confirm the function of dCas9, we constructed a plasmid knocking down <i>lacZ</i>.<br><br />
プラスミドについて書く<br><br />
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<a href="#">Part link</a><br><br><br />
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<h3 style="background-color:#f0ffff ">2.2.Evaluation of <i>Fur</i>,<i>fie</i>F knockdown</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We performed two experiments about CRISPRi system in order to confirm the knockdown function as desired. The purpose is a function check for <i>lacZ</i>, <i>Fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. <br><br />
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<center><img src="https://static.igem.org/mediawiki/2013/7/7f/Chiba.CRISPRi.kan.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 1</b> the method of function check about <i>fur</i></p></center><br />
<br><img src="https://static.igem.org/mediawiki/2013/f/f8/Uptake_image.PNG" width="723px"height="305px"><br><br><br />
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<a href="https://2013.igem.org/Team:Chiba/Assay/uptake">Assay</a><br />
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<h3 style="background-color:#f0ffff ">2.3.Evaluation of absorbed iron volume</h3><br />
<p><br />
<b>Experiment:</b><br><br />
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<left><img src="https://static.igem.org/mediawiki/2013/3/3f/CRISPRi.PNG" width="409px"height="88px"></left><br />
<right><img src="https://static.igem.org/mediawiki/2013/2/23/DCas9.PNG" width="306px"height="110px"></right><br><br />
<center><b>Fig. 2</b></center><br />
<br><p>&nbsp;&nbsp;&nbsp;&nbsp;E. coli stain BL21 was transformed by Plasmid shown in <b>Fig. 2</b> .&nbsp;Then we cultured all transformants with atC.&nbsp; atC was added to knock down <i>fur</i> and <i>fie</i>F.&nbsp; After that we cultured it in the presence of ferric citrate, and measured the density of iron that weren’t taken in to <i>E. coli</i> and remained in the medium by measuring Absorbance (Abs 756) with color reagent(Fig. 2).</p><br />
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<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<p><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid建設の説明</h3><br />
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<h3 style="background-color:#f0ffff ">3.2.Function Check</h3><br />
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<center><img src="https://static.igem.org/mediawiki/2013/6/63/Chiba.CRISPRi.lacZ.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 3</b> Function check about CRISPRi-<i>lacZ</i></p></center><br><br />
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&nbsp;&nbsp;&nbsp;&nbsp;aTcがないときCRISPRi-lacZは発現されないためlacZの抑制は起こらず,X-galは分解されて培地は青く染まった。一方,aTcがあるとCRISPRi-lacZによる抑制が起こるため,lacZは発現されず,培地は白いままとなった。このことから,CRISPRi-lacZの機能を確認することができた。<br><br />
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&nbsp;&nbsp;&nbsp;&nbsp;一方,fur, fieF, trxB, およびgor遺伝子を狙ったgRNAについて,その標的配列の下流にカナマイシン遺伝子を挿入した大腸菌株の作製を試みた。すると,furおよびtrxBの下流にカナマイシン遺伝子を挿入した株(それぞれMG1655Δfur::kmrおよびMG1655ΔtrxB::kmr)が得られた。一方,fieFおよびgorの下流にカナマイシン遺伝子を挿入した株(それぞれMG1655ΔfieF::kmrおよびMG1655Δgor::kmr)は得られなかった。これは,trxBおよびgorのプロモータが弱く,カナマイシン耐性遺伝子の発現量が,大腸菌にカナマイシン耐性を与えるほど多くないためと考えられる。<br><br />
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<h3 style="background-color:#f0ffff ">3.3.鉄取り込み量の評価</h3><br />
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<center><img src="https://static.igem.org/mediawiki/2013/d/d1/Abs_BL.png"width="800px"></center><br><br />
<center>Fig. 4 BL21での、各プラスミドを導入した大腸菌の鉄試薬別のabsorbance</center><br><br><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/8/85/Chiba_Abs_SH.png"width="800px"></center><br><br />
<center>Fig. 5 SHuffle®での、各プラスミドを導入した大腸菌の鉄試薬別のabsorbance</center><br><br />
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BL21、Shuffleともに、各細胞数において、鉄溶液の種類にかかわらず、吸光度にばらつきがでた。<br><br />
これより、この実験方法では、大腸菌の取り込んだ鉄量が少なすぎるため、定量できなかったと考えられる。<br><br />
今後の課題としては、さらに多い細胞数での定量、または、加える鉄試薬の濃度をさらに調整しなおす必要がある。<br><br />
また、細胞破砕によって大腸内の鉄量を定量する方法も検討する必要がある。<br><br />
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<h3 style="background-color:#ffdead ">4.Conclusion</h3><br />
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それは・・<br />
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</html></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/storeTeam:Chiba/Project/store2013-09-27T22:28:28Z<p>T.Senda: </p>
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<h2 id="storage" style="background-color:#ff9933"><center>Sequestration: Fe-storage machine</center></h2><br />
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<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
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<br>&nbsp;&nbsp;&nbsp;&nbsp; <p><b>Fe must be isolated</b>: In order to magnetize <i>E. coli</i>, we need to stuff as much Fe ions as possible in <i>E. coli</i>. Fe(II) could cause Fenton reaction in response to hydrogen peroxide, harmful hydroxyl radicals (OH・). Dilemma is that, feeding too much Fe into cell would kills the host cell. We decided to over express the ferritins that capture and store Fe irons.</p> <br><br />
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<p><b>Fe container machinery</b>: Ferritin particles are made of two small protein subunits (heavy chain (FTH) and light chain (FTL)) (Fig. 1). When expressed, the ferritin subunits automatically assemble into 24-membered protein cages.</p><br />
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<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ferrittin.png"alt=""align="middle"></center><br />
<center>Fig. 1. Complex structure of ferritin<br></center><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Heavy chain catalyzes oxidation of iron and stimulate 2Fe(II)+O2→[Fe(III)-O-O-Fe(III)] reaction<br />
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2Fe<sub>2</sub>+O<sub>2</sub>+(H<sub>2</sub>O)x+3→Fe<sub>2</sub>O<sub>2</sub>(H<sub>2</sub>O)x+4H+H<sub>2</sub>O<sub>2</sub><br />
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<br><br>&nbsp;&nbsp;&nbsp;&nbsp;On the other hand, light chain takes up Fe(III). By functional expression of these two polypeptides, virtual concentration of Fe inside cell can be much reduced. <br />
Interestingly, the storage capacity, the complex size, and FTH/FTL ratio can vary from species to species. Generally speaking, the mammalian ferritin complex contains more FTL than FTH, while the ferritin complex from bacteria have reversed compositions. <br />
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<b>Choice of ferritin</b>: Because the storage capacity of <i>E. coli</i> ferritin is far less than that of mammarian type, we decided to make BioBrick for the functional expression of human ferritin in <i>E. coli</i>.<br />
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<b>Hypothesis</b>: By storing Fe in isolation, the maximum capacity for Fe storage should be elevated. Also, the apparent iron tolerance of <i>E. coli</i> should be also elevated. <br />
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<h3 style="background-color:#ffdead ">2.Experiments</h3><br />
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<h3 style="background-color:#f0ffff ">2.1.BioBrick construction</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;The ratio of FTH/FTL can be flexible in ferritin complex, and there exist a best composition that gives the highest Fe-storage activity. In heterologous expression of ferritin, the translational efficiency can be fine-tuned so that we could achieve that best composition. So, we constructed BioBricks for the functional expression of human ferritin complex in two formats; <br />
<br>BBa_K1057002: 'middle' RBS assigned for FTH <br />
<br>BBa_K1057009: 'strong' RBS assigned for FTH <br />
<br>In both construct, two ferritin genes (FTH and FTL) are placed under pBAD promoter to control the timing and expression level of these genes. To facilitate this construction process, we modified an existing Biobrick (BBa_I74608 deposited by iGEM 2007 team Cambridge) into the new BioBrick(BBa_K1057012). This enabled us the rapid, in-parallel, and one-pot digestion/ ligation using Golden gate method. <br />
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<center><img src="https://static.igem.org/mediawiki/2013/3/32/Chiba.ferritin.png"alt=""align="middle"></center><br />
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<center><p>Fig. 2. Cloning procedure of ferritin-producing BioBrick</p></center><br><br />
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<a href="https://2013.igem.org/Team:Chiba/Parts"> Parts link</a><br />
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<h3 style="background-color:#f0ffff ">2.2.Expression of Ferritin in protein level</h3><br />
<p><br />
Our new BioBricks BBa_K1057002 and BBa_K1057009 were transfected into in BL21 strain. The expression level of each individual components (H-chain and L-chain) was checked by SDS-PAGE/ coomassie blue. As a control, we also conducted the same experiment with “sfgfp generator”.<br />
<br><br />
The results are shown in Fig. 3. Both in total protein fraction (gel in left) and soluble fraction(gel in right), there observed two characteristic bands: one was with the size of 20 kDa (corresonding to FTH), while the other was 19kDa (corresponding to FTL). These bands were not detectable for the control sample (Lane 3) expressing the control gene sfGFP under the control of pBAD promoter.<br />
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<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ike.png" width="750px"height="500px"></center><br><br />
<center><p>Fig. 3. Expression of ferritin in <i>E. coli</i> treated with arabinose. <br />
<br> lane 1. pBAD/araC-ferritin-strong(BBa_K1057009)<br />
<br>lane 2. pBAD/araC-ferritin-mid(BBA_K1057002)<br />
<br>lane 3. BBa_I746908(sfgfp)<br />
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<h3 style="background-color:#f0ffff ">2.3.Evaluation of iron tolerance</h3><br />
<p><br />
<a href="https://2013.igem.org/Team:Chiba/Assay/store">Assay</a><br><br />
Experiment: We constructed two kinds of plasmids with which RBS scores are different as shown in Fig. 2. E. coli strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with above plasmids. Human ferritin genes (fth1 and ftl) are placed on the high-copy plasmid under the control of BAD promoter. To express Human ferritin proteins, arabinose was added into media(final conc. 0.2%). The resultant “ferritin generators” were cultured in the presence of iron citrate (Fe3+) or iron ascorbate (Fe2+), and checked final cell density and colony forming efficiency. As a control, we conducted the same experiment with “sfgfp generator”. <br />
</p><br />
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<h3 style="background-color:#f0ffff ">2.4.Evaluation of magnetism</h3><br />
<p><br />
We examine whether BL21 overexpressing ferritin is attracted to a magnet.<br><br />
Our experimental setup is shown below(Fig. 3).<br><br />
<br />
The details are shown in <a href="https://2013.igem.org/Team:Chiba/Assay/store">assay.</a><br><br />
If <i>E. coli</i> have magnetism, the attract to magnets!<br><br />
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<center><img src="https://static.igem.org/mediawiki/2013/e/e5/Chiba_magnet_method_ssp.png" width="600px"height="400px"></center><br><br />
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<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid</h3><br />
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<h3 style="background-color:#f0ffff ">3.2 Assessment of iron tolerance</h3><br />
<br><p><b>Result. 1</b><br />
<br><left><img src="https://static.igem.org/mediawiki/2013/7/7f/Strorage_BL21_asc_OD_cor.PNG" width="377px"height="289px"></left><br />
<right><img src="https://static.igem.org/mediawiki/2013/f/f5/Storage_SHuffle_asc_OD_cor.PNG" width="372px"height="288px"></right><br><br />
<br><center>Fig. 4</center><br />
<br><br />
<br><p>Fig. 4 shows the optical density of <i>E. coli</i> which is cultured in the presence of iron for 12h (at 37°C).<br><br />
(Approximately 10^7 cells inoculated in to fresh media(2 mL, containing iron)in each.)<br />
<br>・Overexpression of ferritin didn’t affect the growth so much.<br />
<br>・The one without overexpression of ferritin, growth inhibition was observed when the concentration of ferrous ascorbate is 6-8mM. And when the concentration of ferrous ascorbate was over 8 mM, the growth of <i>E. coli</i> wasn’t observed at all.</p><br />
<br>・一方,フェリチンを過剰発現した大腸菌は,アスコルビン酸鉄濃度5-7 mMの範囲では,アスコルビン酸鉄不在下よりも増殖がよく?~ちょっとここ考えます,また9-10 mMのアスコルビン酸鉄濃度においても,細胞は増殖した<br />
<br>・<i>E. coli</i> strain <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> showed same results.<br />
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<br><br><b>Result. 2</b><br />
<br><center><img src="https://static.igem.org/mediawiki/2013/3/39/Storage_result_BL_asc.PNG" width="716px"height="299px"></center><br><br />
<br>The ability to form colony changed similarly.<br />
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<h3 style="background-color:#f0ffff ">3.3 Evaluation of magnetism</h3><br />
<p><b>Result</b><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/2a/Chiba_tetsu_BL21_str_no2.png" width="571px"height="500px"></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/1/10/Chiba_tetsu_BL21_mid_no2.png" width="571px"height="500px"></center><br><br />
<p>Each <i>E. coli</i> don`t move at all.<br><br />
We consider this cause is uptake quantity of Fe(Ⅲ) is not sufficiently.<br><br />
So, expression levels of ferritin in each <i>E. coli</i> is not sufficiently.<br><br />
Future subject is to increase uptake quantity of Fe(Ⅲ) to knock down/out <i>Fur</i> and <i>fie</i>F.</p><br><br />
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<h3 style="background-color:#ffdead ">4.Conclusion</h3><br />
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<p><br />
それは・・<br />
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</html></div>T.Sendahttp://2013.igem.org/File:Chiba_ike.pngFile:Chiba ike.png2013-09-27T22:24:56Z<p>T.Senda: </p>
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<div></div>T.Sendahttp://2013.igem.org/Team:Chiba/PartsTeam:Chiba/Parts2013-09-27T22:07:10Z<p>T.Senda: </p>
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<p><center><groupparts>iGEM013 Chiba</groupparts></center></p><br />
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<h2 style="background-color:#ff9933 ">Ferritin</h2><br />
<h3 style="background-color:#ffdead ">1. Introduction</h3><br />
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<h2 style="background-color:#ff9933">CRISPRi</h2><br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<p><br />
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&nbsp;&nbsp;&nbsp;&nbsp;One of the immune system is CRISPR (clustered regularly interspaced short palindromic repeats). Cas9 protein and sgRNA (small guide RNA) combine specific sequence and cut it. Using a modified Cas9 lacking endonucleolytic activity, we can use CRISPR as repressor. This system is CRISPRi (CRISPR interference) as shown in Fig. 1 Designing guide region of sgRNA and coexpress dCas9, you can knockdown target gene conditionally.<br><br />
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<center><img src="https://static.igem.org/mediawiki/2013/b/bc/Chiba.CRISPRi.gaiyo.png"alt=""align="middle"></center><br><br />
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<center>Figure n. CRISPRi mechanism</center><br><br />
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<h2 style="background-color:#ff9933 ">Improvement Parts : expression vector with pBAD/Ara switch compatible for<br />
"Golden gate gene swapping"</h2><br />
<h3 style="background-color:#ffdead ">1. Introduction</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp; Gentic switch such as pBAD/araC system is very useful for overexpression of given genes. In order to place the various open reading frames with its RBS under the pBAD/araC system, we improved BBa_I74608 to insert BsaI site in both sides of sfgfp gene. This improvement enables us to use ‘Golden Gate’ cloning Method as described below (Fig. 2):<br><br />
1) Preparation of insert fragment : Given gene(s) are PCR amplified with the additional sequence coding for ribosome-binding sites and BsaI site. <br><br />
2) BBa_I74608 and PCR amplified insert fragment is BsaI digested and ligated in a single-pot reaction.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;This method is designed BsaI site doesn't remain on the vector after digesting BsaI. So, you can perform digestion and ligation at the same time. You can obtain desired plasmids in a short time.<br><br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/b/be/Chiba.goldengate.png"alt=""align="middle"></center><br><br />
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<center><p>Fig. 1 Golden Gate cloning strategy</center></p><br> <br />
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</p><br />
<h3 style="background-color:#ffdead ">2. Material & Method</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We performed Golden Gate cloning with this part (vector) and mRFP (insert) and checked function. And we investigated the reaction rate changing mol ratio of vector to insert. The protocol is below.<br><br />
1) PCR up insert with BsaI site<br><br />
2) Golden Gate cloning<br><br />
3) transformation<br><br />
Mixture list in Golden Gate is below.<br><br />
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<br />
</p><br />
<h3 style="background-color:#ffdead ">3. Result</h3><br />
<p><br />
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<br><br />
<center><p>Table 1 cfu/transformation</p></center><br />
<center><img src="https://static.igem.org/mediawiki/2013/0/0e/Chiba.goldengate.cfu.png"alt=""align="middle"></center><br><br />
<center><p>Table 2 Reaction ratio</p></center><br />
<center><img src="https://static.igem.org/mediawiki/2013/1/15/Chiba.goldengate.reactionrate.png"alt=""align="middle"></center><br><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/1/13/Chiba.goldengate.reactionrate.graph.png"alt=""align="middle"></center><br />
<center><p>Fig. 2 Reaction ratio (N.D.: Not Detected)</p></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/9/92/Chiba.goldengate.plate.png"alt=""align="middle"></center><br />
<center><p>Fig. 3 Plate</p></center><br><br />
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<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;In the traditional ligation, the best ratio is vecor: insert= 1: 3. However, according to this experiment, the best ratio was vector: insert= 1: 1in the Golden Gate. The vector ............................... vectorが切られたあと再びsfGFPとligateする可能性もあり,これがまた切られるためにBsaIが使用される。したがって,insertが多いとvectorとBsaIの衝突頻度が低下するため,liationが進みにくいと考えられる。<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;The maximum reaction rate was 68.9%. There existed few back ligations. Therefore, selecting colonies not shining green, you can pick the desired colonies easily.<br><br />
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</html></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/uptakeTeam:Chiba/Project/uptake2013-09-27T21:55:37Z<p>T.Senda: </p>
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<h2 id="uptake" style="background-color:#ff9933"><center>Uptake</center></h2><br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
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<p><br />
<h3 style="background-color:#f0ffff ">1.1.<i>Fur</i></h3><br />
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&nbsp;&nbsp;&nbsp;&nbsp;<p><i>Fur</i> (Ferric uptake regulator) controls iron metabolism such as taking iron in or storing iron.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In most cases, it combines with <i>Fur</i> box (which is near the promoter) and regulates transcription.</p><br />
&nbsp;&nbsp;&nbsp;&nbsp;<p>When iron is rich, <i>Fur</i> becomes active and when <i>Fur</i> becomes active, it restricts the expression of iron transporter, and that means that the iron uptake would stop. At the same time,<i> Fur</i> restricts the expression of <i>Ryh</i>B. <i>RyhB</i> is one of the sRNA that restricts the expression of Ferritin, so making <i>Fur</i> active leads ferritin to express.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Conversely, when iron is lacking, <i>Fur</i> becomes inactive, and the iron transporter would work normally, so iron would be taken in. <i>Ryh</i>B would also work normally, so expression of ferritin is restricted and Ferritin wouldn't be expressed.</p><br><br />
&nbsp;&nbsp;&nbsp;&nbsp;<p>In short, if we knockdown/knockout <i>Fur</i>, iron transporter would be active so iron would be taken in, but the expression of Ferritin is stopped by <i>Ryh</i>B.</p><br><br />
<br><br />
<h3 style="background-color:#f0ffff ">1.2.<i>fie</i>F</h3><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;<p>There is a Regulator called <i>fie</i>F (ferric iron efflux). It makes iron and zinc flow out of cell and controls detoxification of cell. When <i>fie</i>F is knocked down/out, the tolerance of cell to iron would be lowered.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;<i>Fec</i> has a character that can recognize ferric citrate and taking Fe(III) in. <i>Fec</i> is controlled by <i>Fur</i> and when the density of Fe(III) in the cell is too high, <i>Fur</i> restricts Fec and stops iron uptake.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;In the same way, <i>Efe</i>UOB has a character that can recognize ascorbic acid and taking Fe(II) in. <i>Efe</i>UOB is also controlled by <i>Fur</i> and when the density of Fe(II) in the cell is too high, <i>Fur</i> restricts <i>EfeUOB</i> and stops iron uptake.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Using these characters, and by knocking down/out <i>Fur</i> and <i>fie</i>F, <i>Fec</i>/<i>Efe</i>UOB expression wouldn't be restricted so the amount of iron coming in would increase and the amount of iron going out would decrease. The system would work like when iron is lacking.<br><br />
<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;As a result, the amount of iron inside <i>E. coli</i> would increase.</p><br />
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</p><br />
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<h3 style="background-color:#ffdead ">2.Materials&Methods</h3><br />
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<h3 style="background-color:#f0ffff ">2.1.plasmid construct</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp;We constructed four plasmids knocking down <i>fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. at the same time, in order to confirm the function of dCas9, we constructed a plasmid knocking down <i>lacZ</i>.<br><br />
プラスミドについて書く<br><br />
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<a href="#">Part link</a><br><br><br />
</p><br />
<h3 style="background-color:#f0ffff ">2.2.Evaluation of <i>Fur</i>,<i>fie</i>F knockdown</h3><br />
<p><br />
We performed two experiments about CRISPRi system in order to confirm the knockdown function as desired. The purpose is a function check for <i>lacZ</i>, <i>Fur</i>, <i>fie</i>F, <i>gor</i>, and <i>trx</i>B. <br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/7/7f/Chiba.CRISPRi.kan.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 1</b> the method of function check about <i>fur</i></p></center><br />
<br><img src="https://static.igem.org/mediawiki/2013/f/f8/Uptake_image.PNG" width="723px"height="305px"><br><br><br />
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<a href="https://2013.igem.org/Team:Chiba/Assay/uptake">Assay</a><br />
</p><br />
<h3 style="background-color:#f0ffff ">2.3.Evaluation of absorbed iron volume</h3><br />
<p><br />
<b>Experiment:</b><br><br />
<br />
<br />
<left><img src="https://static.igem.org/mediawiki/2013/3/3f/CRISPRi.PNG" width="409px"height="88px"></left><br />
<right><img src="https://static.igem.org/mediawiki/2013/2/23/DCas9.PNG" width="306px"height="110px"></right><br><br />
<center><b>Fig. 2</b></center><br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;<p>E. coli stain BL21 was transformed by Plasmid shown in <b>Fig. 2</b> .&nbsp;Then we cultured all transformants with atC.&nbsp; atC was added to knock down <i>fur</i> and <i>fie</i>F.&nbsp; After that we cultured it in the presence of ferric citrate, and measured the density of iron that weren’t taken in to <i>E. coli</i> and remained in the medium by measuring Absorbance (Abs 756) with color reagent(Fig. 2).</p><br />
</p><br />
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<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<p><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid建設の説明</h3><br />
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<h3 style="background-color:#f0ffff ">3.2.Function Check</h3><br />
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<center><img src="https://static.igem.org/mediawiki/2013/6/63/Chiba.CRISPRi.lacZ.png"alt=""align="middle"></center><br><br />
<center><p><b>Fig. 3</b> Function check about CRISPRi-<i>lacZ</i></p></center><br><br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp;aTcがないときCRISPRi-lacZは発現されないためlacZの抑制は起こらず,X-galは分解されて培地は青く染まった。一方,aTcがあるとCRISPRi-lacZによる抑制が起こるため,lacZは発現されず,培地は白いままとなった。このことから,CRISPRi-lacZの機能を確認することができた。<br><br />
<br />
&nbsp;&nbsp;&nbsp;&nbsp;一方,fur, fieF, trxB, およびgor遺伝子を狙ったgRNAについて,その標的配列の下流にカナマイシン遺伝子を挿入した大腸菌株の作製を試みた。すると,furおよびtrxBの下流にカナマイシン遺伝子を挿入した株(それぞれMG1655Δfur::kmrおよびMG1655ΔtrxB::kmr)が得られた。一方,fieFおよびgorの下流にカナマイシン遺伝子を挿入した株(それぞれMG1655ΔfieF::kmrおよびMG1655Δgor::kmr)は得られなかった。これは,trxBおよびgorのプロモータが弱く,カナマイシン耐性遺伝子の発現量が,大腸菌にカナマイシン耐性を与えるほど多くないためと考えられる。<br><br />
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<h3 style="background-color:#f0ffff ">3.3.鉄取り込み量の評価</h3><br />
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<center><img src="https://static.igem.org/mediawiki/2013/d/d1/Abs_BL.png"width="800px"></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/8/85/Chiba_Abs_SH.png"width="800px"></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/e/e0/Chiba_Kenryo.png"width="800px"></center><br><br />
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<h3 style="background-color:#ffdead ">4.Conclusion</h3><br />
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それは・・<br />
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</html></div>T.Sendahttp://2013.igem.org/File:Chiba_Kenryo.pngFile:Chiba Kenryo.png2013-09-27T21:54:38Z<p>T.Senda: </p>
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<div></div>T.Sendahttp://2013.igem.org/File:Abs_BL.pngFile:Abs BL.png2013-09-27T21:47:28Z<p>T.Senda: </p>
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<div></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/storeTeam:Chiba/Project/store2013-09-27T20:23:51Z<p>T.Senda: </p>
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<h2 id="storage" style="background-color:#ff9933"><center>Sequestration: Fe-storage machine</center></h2><br />
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<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
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<br>&nbsp;&nbsp;&nbsp;&nbsp; In order to magnetize <i>E. coli</i>, we need to stuff as much Fe ions as possible in <i>E. coli</i>. However, too much Fe would kills the host cell. As a result, giving iron to <i>E.coli</i> excessively leads to the death of <i>E.coli</i>.<br />
<br>We decided to over express the ferritins that capture and store Fe irons. Ferritins form 24-membered protein cages composed by two small proteins called heavy chain (FTH) and light chain (FTL) (Fig. 1). <br><br />
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<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ferrittin.png"alt=""align="middle"></center><br />
<center>Fig. 1. structure of ferritin<br></center><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Fe(II) in <i>E.coli</i> causes Fenton reaction in response to hydrogen peroxide and produce hydroxyl radical (OH・) which is harmful to <i>E.coli</i>. <br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;Ferritin is an intracellular protein which has a property to store iron. Ferritin has Heavy chain and Light chain. Heavy chain affects the oxidation of iron and stimulate 2Fe(II)+O<sub>2</sub>→[Fe(III)-O-O-Fe(III)] reaction. Light chain takes in Fe(III) in ferritin. Fe<sub>2</sub>O<sub>3</sub>(H<sub>2</sub>O) has paramagnetism.<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;These two effects enable isolation of iron in ferritin and enhance <i>E.coli</i> iron tolerance.<br />
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The H chain oxidizes Fe, while L chain stores it. The reaction catalyzed by H chain is shown below.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2Fe<sub>2</sub>+O<sub>2</sub>+(H<sub>2</sub>O)x+3→Fe<sub>2</sub>O<sub>2</sub>(H<sub>2</sub>O)x+4H+H<sub>2</sub>O<sub>2</sub><br><br />
From species to species, the complex size, as well as the FTH/FTL ratio varies. Generally, the mammalian ferritin complex contains more FTL than FTH, while the ferritin complex from bacteria have reversed compositions. Because the storage capacity of <i>E. coli</i> ferritin is far less than that of mammarian type. Therefore, we decided to make BioBrick for the functional expression of human ferritin <i>E.coli</i>.<br><br />
</br><br />
</br></br><br />
&nbsp;&nbsp;&nbsp;&nbsp;Therefore, value of intracellular iron should increase. Also, Cell death may be difficult to occur because of decreasing Fe(ii) concetration.<br />
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<h3 style="background-color:#ffdead ">2.Materials & Methods</h3><br />
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<h3 style="background-color:#f0ffff ">2.1.Parts</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp; The ferritin has FTH and FTL but the ratio is different in the species. However, it may be depend on the expression. So, we constructed two different human ferritin; BBa_K1057002 with middle RBS and BBa_K1057009 with strong RBS. Further more, we placed two ferritin genes (FTH and FTL) under pBAD promoter to control the expression level ant the timing. For these reasons, we constructed a new BioBrick based on BBa_I74608 (deposited by iGEM 2007 team Cambridge). <br><br />
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<center><img src="https://static.igem.org/mediawiki/2013/3/32/Chiba.ferritin.png"alt=""align="middle"></center><br />
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<center><p>Fig. 2. Cloning of ferritin</p></center><br><br />
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<a href="https://2013.igem.org/Team:Chiba/Parts"> Parts link</a><br />
</p><br />
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<br />
<h3 style="background-color:#f0ffff ">2.2.Expression check</h3><br />
<p><br />
pBAD/araC-ferritin-strong and pBAD/araC-ferritin-mid were expressed individually in BL21 strain.<br><br />
We performed SDS-PAGE to check the expression of pBAD/araC-ferritin-strong and pBAD/araC-ferritin-mid.<br><br />
As a control, we conducted the same experiment with “sfgfp generator”.<br><br />
The results of SDS-PAGE were shown below.<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/e/e6/Chiba_SDS_no3s.png" width="750px"height="500px"></center><br><br />
<center>Fig. 3:SDS-PAGE patterns of proteins extracted from <i>E. coli</i> introduced pBAD/araC-ferritin-strong, pBAD/araC-ferritin-mid, or BBa_I746908(sfgfp): (1)pBAD/araC-ferritin-strong, (2)pBAD/araC-ferritin-mid, (3)BBa_I746908(sfgfp)</center>. Left is the result of all fraction, and right is soluble fraction. There exist 2 bands around 20 kDa in both pBAD/araC-ferritin-strong and pBAD/araC-ferritin-mid. The band of 20 kDa is FTH. Another band, the band of 19 kDa, is FTL. There does not exist such bands in Lane(3), but exist a different band in 30 kDa. This is a band of sfgfp.<br />
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</p><br />
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<h3 style="background-color:#f0ffff ">2.3.鉄耐性の評価</h3><br />
<p><br />
<a href="https://2013.igem.org/Team:Chiba/Assay/store">Assay</a><br><br />
Experiment: We constructed two kinds of plasmids with which RBS scores are different as shown in Fig. 2. E. coli strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with above plasmids. Human ferritin genes (fth1 and ftl) are placed on the high-copy plasmid under the control of BAD promoter. To express Human ferritin proteins, arabinose was added into media(final conc. 0.2%). The resultant “ferritin generators” were cultured in the presence of iron citrate (Fe3+) or iron ascorbate (Fe2+), and checked final cell density and colony forming efficiency. As a control, we conducted the same experiment with “sfgfp generator”. <br />
</p><br />
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<h3 style="background-color:#f0ffff ">2.4.Evaluation of magnetism</h3><br />
<p><br />
We examine whether BL21 overexpressing ferritin is attracted to a magnet.<br><br />
Our experimental setup is shown below(Fig. 3).<br><br />
<br />
The details are shown in <a href="https://2013.igem.org/Team:Chiba/Assay/store">assay.</a><br><br />
If <i>E. coli</i> have magnetism, the attract to magnets!<br><br />
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<center><img src="https://static.igem.org/mediawiki/2013/e/e5/Chiba_magnet_method_ssp.png" width="600px"height="400px"></center><br><br />
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</p><br />
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<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid</h3><br />
<br />
<h3 style="background-color:#f0ffff ">3.2 Assessment of iron tolerance</h3><br />
<br><p><b>Result. 1</b><br />
<br><left><img src="https://static.igem.org/mediawiki/2013/7/7f/Strorage_BL21_asc_OD_cor.PNG" width="377px"height="289px"></left><br />
<right><img src="https://static.igem.org/mediawiki/2013/f/f5/Storage_SHuffle_asc_OD_cor.PNG" width="372px"height="288px"></right><br><br />
<br><center>fig.A</center><br />
<br><br />
<br>Fig. A shows the optical density of <i>E. coli</i> which is cultured in the presence of iron for 12h (at 37°C).<br><br />
(Approximately 10^7 cells inoculated in to fresh media(2 mL, containing iron)in each.)<br />
<br>・Overexpression of ferritin didn’t affect the growth so much.<br />
<br>・The one without overexpression of ferritin, growth inhibition was observed when the concentration of ferrous ascorbate is 6-8mM. And when the concentration of ferrous ascorbate was over 8 mM, the growth of <i>E. coli</i> wasn’t observed at all.<br />
<br>・一方,フェリチンを過剰発現した大腸菌は,アスコルビン酸鉄濃度5-7 mMの範囲では,アスコルビン酸鉄不在下よりも増殖がよく?~ちょっとここ考えます,また9-10 mMのアスコルビン酸鉄濃度においても,細胞は増殖した<br />
<br>・<i>E. coli</i> strain <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> showed same results.<br />
<br />
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<br><br><b>Result. 2</b><br />
<br><center><img src="https://static.igem.org/mediawiki/2013/3/39/Storage_result_BL_asc.PNG" width="716px"height="299px"></center><br><br />
<br>The ability to form colony changed similarly.<br />
<br><br />
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<h3 style="background-color:#f0ffff ">3.3磁性評価</h3><br />
<p><b>Result</b><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/2a/Chiba_tetsu_BL21_str_no2.png" width="571px"height="500px"></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/1/10/Chiba_tetsu_BL21_mid_no2.png" width="571px"height="500px"></center><br><br />
Each <i>E. coli</i> don`t move at all.<br><br />
We consider this cause is uptake quantity of Fe(Ⅲ) is not sufficiently.<br><br />
So, expression levels of ferritin in each <i>E. coli</i> is not sufficiently.<br><br />
Future subject is to increase uptake quantity of Fe(Ⅲ) to knock down/out <i>Fur</i> and <i>fie</i>F.<br><br />
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</p><br />
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<h3 style="background-color:#ffdead ">4.Conclusion</h3><br />
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<p><br />
それは・・<br />
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</html></div>T.Sendahttp://2013.igem.org/Team:Chiba/NotebookTeam:Chiba/Notebook2013-09-27T20:21:41Z<p>T.Senda: </p>
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.article h3 a {<br />
display:block;<br />
font-color:#3cb371;<br />
<br />
}<br />
<br />
<br />
.article h3 +div {<br />
max-height:0;<br />
overflow:hidden;<br />
-webkit-transition:max-height 1s ;<br />
transition:max-height 1s;<br />
}<br />
.article h3:target +div {<br />
max-height:1000px;<br />
overflow:auto;<br />
margin:5px;<br />
}<br />
<br />
<br />
p{font-size:12px;<br />
font-color: #808080;<br />
}<br />
<br />
a{color:#006400;}<br />
<br />
<br />
</style><br />
</head><br />
<br />
<br />
<body><br />
<div class="textSpace"><br />
<br />
<br />
<div class="section"><br />
<br />
<div class="article"><br />
<h3 id="c1"><a href="#c1">week1(8/18/2013~8/25/2013)</a></h3><br />
<div><br />
<p><br />
<b>8/22//2013</b><br><br />
.Extracting the human ferritin plasmid and iron chaperone plasmid<br><br />
<b>8/23/2013</b><br><br />
.BBa_I746908 and BBa_I746902 transformation<br><br />
<b>8/24/2013</b><br><br />
.Extracting BBa_I746908 and BBa_I746902<br><br />
<b>8/25/2013</b><br><br />
<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="article"><br />
<h3 id="c2"><a href="#c2">week2(8/26/2013~9/1/2013)</a></h3><br />
<div><br />
<p><br />
<b>8/26/2013</b><br><br />
<br />
<b>8/27/2013</b><br><br />
<br />
<b>8/28/2013</b><br><br />
<br />
<b>8/29/2013</b><br><br />
.Gel electrophoresis(ferritin,iron chaperone4-6c,4-14I)<br><br />
.PCR:(4-6c),ferritin(mid,strong)<br><br />
.Digestion test:4-6c,ferritin,iron chaperone<br><br />
.Pre-culture:CRISPRi<br><br />
<br />
<br />
<b>8/30/2013</b><br><br />
.Extracting CRISPRi plasmids(fur,fieF,gor,and trxB)<br><br />
.PCR:ferritin(min,strong),CRISPRi(fur,fieF,for,and trxB)<br><br />
.Gel electrophresis:PCR products<br><br />
.Gel extractions:PCR products<br><br />
<b>8/31/2013</b><br><br />
.Digestion:ferritin(mid and strong),4-6c<br><br />
<br />
<b>9/1/2013</b><br><br />
.FASTR:CRISPRi<br><br />
.Ligation:ferritin and 4-6c<br><br />
.Begining editing Wiki<br><br />
<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="article"><br />
<h3 id="c3"><a href="#c3">week3(9/2/2013~9/7/2013)</a></h3><br />
<div><br />
<p><br />
<b>9/2/2013</b><br><br />
.Making plates with Broth and methylene blue(MB)<br><br />
<b>9/3/2013</b><br><br />
.Making olates with Broth and MB<br><br />
.Culturing BL21 and shuffle , respectively<br><br />
<b>9/4/2013</b><br><br />
<br />
<b>9/5/2013</b><br><br />
<br />
<b>9/6/2013</b><br><br />
<br />
<b>9/7/2013</b><br><br />
<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="article"><br />
<h3 id="c4"><a href="#c4">week4(9/8/2013~9/14/2013)</a></h3><br />
<div><br />
<p><br />
<br />
<b>9/9/2013</b><br><br />
.Hold a presentation(state of progress)<br><br />
.<br><br />
<b>9/10/2013</b><br><br />
<br />
<b>9/11/2013</b><br><br />
<br />
<b>9/12/2013</b><br><br />
<br />
<b>9/13/2013</b><br><br />
<br />
<b>9/14/2013</b><br><br />
<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br />
<br />
<h2 style="background-color:#ff9933"><center>Assay</center></h2><br />
<br />
<center><br />
<table border="1"> <br />
<br />
<br />
<tr><br />
<br />
<td align="center"><br />
<a href="https://2013.igem.org/Team:Chiba/Assay/uptake"><img src = "https://static.igem.org/mediawiki/2013/archive/c/c2/20130927162646%21Chiba_uptake.jpg" ALT = "#"width="216.5px"height="349px"></A><br />
</td><br />
<br />
<br />
<td align="center"><br />
<a href="https://2013.igem.org/Team:Chiba/Assay/store"><img src = "https://static.igem.org/mediawiki/2013/a/a3/Chiba_storage.jpg" ALT = "#"width="211px"height="346.5px"></a><br />
</td><br />
<br />
<td align="center"><br />
<a href="https://2013.igem.org/Team:Chiba/Assay/oxidation"><img src = "https://static.igem.org/mediawiki/2013/archive/8/8b/20130927163941%21Chiba_oxidation.jpg" ALT = "#"width="218px"height="349.5px"></a><br />
</td><br />
<br />
<br />
<br />
</tr><br />
<br />
<tr><br />
<br />
<th><br />
<a href="https://2013.igem.org/Team:Chiba/Project/uptake">Iron Uptake</a><br />
</th><br />
<br />
<th><br />
<a href="https://2013.igem.org/Team:Chiba/Project/store">Iron Storage</a><br />
</th><br />
<br />
<th><br />
<a href="https://2013.igem.org/Team:Chiba/Project/oxidation">Iron Oxidation</a><br />
</th><br />
<br />
</tr><br />
<br />
<br />
<br />
<br />
</table><br />
</center><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</body><br />
</html></div>T.Sendahttp://2013.igem.org/Team:Chiba/NotebookTeam:Chiba/Notebook2013-09-27T20:21:07Z<p>T.Senda: </p>
<hr />
<div>{{Chiba_base}}<br />
<html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html;charset=utf-8"/><br />
<title>iGEM-2013 Chiba</title><br />
<style type="text/css"><br />
<br />
.section{<br />
float:left;<br />
width:700px;<br />
padding-left:50px;<br />
}<br />
<br />
.aside{<br />
float:left;<br />
width:10px<br />
padding-left:50px;<br />
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<br />
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.article {<br />
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border: solid 1px #ccc;<br />
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margin:0 5px;<br />
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.article:last-child {<br />
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.article h3 {<br />
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.article h3 a {<br />
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<br />
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<br />
<br />
.article h3 +div {<br />
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-webkit-transition:max-height 1s ;<br />
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.article h3:target +div {<br />
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<br />
<br />
p{font-size:12px;<br />
font-color: #808080;<br />
}<br />
<br />
a{color:#006400;}<br />
<br />
<br />
</style><br />
</head><br />
<br />
<br />
<body><br />
<div class="textSpace"><br />
<br />
<br />
<div class="section"><br />
<br />
<div class="article"><br />
<h3 id="c1"><a href="#c1">week1(8/18/2013~8/25/2013)</a></h3><br />
<div><br />
<p><br />
<b>8/22//2013</b><br><br />
.Extracting the human ferritin plasmid and iron chaperone plasmid<br><br />
<b>8/23/2013</b><br><br />
.BBa_I746908 and BBa_I746902 transformation<br><br />
<b>8/24/2013</b><br><br />
.Extracting BBa_I746908 and BBa_I746902<br><br />
<b>8/25/2013</b><br><br />
<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="article"><br />
<h3 id="c2"><a href="#c2">week2(8/26/2013~9/1/2013)</a></h3><br />
<div><br />
<p><br />
<b>8/26/2013</b><br><br />
<br />
<b>8/27/2013</b><br><br />
<br />
<b>8/28/2013</b><br><br />
<br />
<b>8/29/2013</b><br><br />
.Gel electrophoresis(ferritin,iron chaperone4-6c,4-14I)<br><br />
.PCR:(4-6c),ferritin(mid,strong)<br><br />
.Digestion test:4-6c,ferritin,iron chaperone<br><br />
.Pre-culture:CRISPRi<br><br />
<br />
<br />
<b>8/30/2013</b><br><br />
.Extracting CRISPRi plasmids(fur,fieF,gor,and trxB)<br><br />
.PCR:ferritin(min,strong),CRISPRi(fur,fieF,for,and trxB)<br><br />
.Gel electrophresis:PCR products<br><br />
.Gel extractions:PCR products<br><br />
<b>8/31/2013</b><br><br />
.Digestion:ferritin(mid and strong),4-6c<br><br />
<br />
<b>9/1/2013</b><br><br />
.FASTR:CRISPRi<br><br />
.Ligation:ferritin and 4-6c<br><br />
.Begining editing Wiki<br><br />
<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="article"><br />
<h3 id="c3"><a href="#c3">week3(9/2/2013~9/7/2013)</a></h3><br />
<div><br />
<p><br />
<b>9/2/2013</b><br><br />
.Making plates with Broth and methylene blue(MB)<br><br />
<b>9/3/2013</b><br><br />
.Making olates with Broth and MB<br><br />
.Culturing BL21 and shuffle , respectively<br><br />
<b>9/4/2013</b><br><br />
<br />
<b>9/5/2013</b><br><br />
<br />
<b>9/6/2013</b><br><br />
<br />
<b>9/7/2013</b><br><br />
<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="article"><br />
<h3 id="c4"><a href="#c4">week4(9/8/2013~9/14/2013)</a></h3><br />
<div><br />
<p><br />
<br />
<b>9/9/2013</b><br><br />
.Hold a presentation(state of progress)<br><br />
.<br><br />
<b>9/10/2013</b><br><br />
<br />
<b>9/11/2013</b><br><br />
<br />
<b>9/12/2013</b><br><br />
<br />
<b>9/13/2013</b><br><br />
<br />
<b>9/14/2013</b><br><br />
<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br />
<br />
<h2 style="background-color:#ff9933"><center>Project</center></h2><br />
<br />
<center><br />
<table border="1"> <br />
<br />
<br />
<tr><br />
<br />
<td align="center"><br />
<a href="https://2013.igem.org/Team:Chiba/Assay/uptake"><img src = "https://static.igem.org/mediawiki/2013/archive/c/c2/20130927162646%21Chiba_uptake.jpg" ALT = "#"width="216.5px"height="349px"></A><br />
</td><br />
<br />
<br />
<td align="center"><br />
<a href="https://2013.igem.org/Team:Chiba/Assay/store"><img src = "https://static.igem.org/mediawiki/2013/a/a3/Chiba_storage.jpg" ALT = "#"width="211px"height="346.5px"></a><br />
</td><br />
<br />
<td align="center"><br />
<a href="https://2013.igem.org/Team:Chiba/Assay/oxidation"><img src = "https://static.igem.org/mediawiki/2013/archive/8/8b/20130927163941%21Chiba_oxidation.jpg" ALT = "#"width="218px"height="349.5px"></a><br />
</td><br />
<br />
<br />
<br />
</tr><br />
<br />
<tr><br />
<br />
<th><br />
<a href="https://2013.igem.org/Team:Chiba/Project/uptake">Iron Uptake</a><br />
</th><br />
<br />
<th><br />
<a href="https://2013.igem.org/Team:Chiba/Project/store">Iron Storage</a><br />
</th><br />
<br />
<th><br />
<a href="https://2013.igem.org/Team:Chiba/Project/oxidation">Iron Oxidation</a><br />
</th><br />
<br />
</tr><br />
<br />
<br />
<br />
<br />
</table><br />
</center><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</body><br />
</html></div>T.Sendahttp://2013.igem.org/Team:Chiba/NotebookTeam:Chiba/Notebook2013-09-27T20:16:45Z<p>T.Senda: </p>
<hr />
<div>{{Chiba_base}}<br />
<html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html;charset=utf-8"/><br />
<title>iGEM-2013 Chiba</title><br />
<style type="text/css"><br />
<br />
.section{<br />
float:left;<br />
width:700px;<br />
padding-left:50px;<br />
}<br />
<br />
.aside{<br />
float:left;<br />
width:10px<br />
padding-left:50px;<br />
}<br />
<br />
<br />
.article {<br />
padding: 5px;<br />
border: solid 1px #ccc;<br />
margin: 10px;<br />
zoom: 1;<br />
font-size:15px;<br />
}<br />
<br />
.article {<br />
border:solid 1px #999;<br />
border-bottom:0;<br />
margin:0 5px;<br />
padding:0;<br />
}<br />
.article:last-child {<br />
border-bottom:solid 1px #999;<br />
}<br />
.article h3 {<br />
background-color: #ffc0cb;<br />
padding-left:4px;<br />
font-color:#006400;<br />
}<br />
.article h3 a {<br />
display:block;<br />
font-color:#3cb371;<br />
<br />
}<br />
<br />
<br />
.article h3 +div {<br />
max-height:0;<br />
overflow:hidden;<br />
-webkit-transition:max-height 1s ;<br />
transition:max-height 1s;<br />
}<br />
.article h3:target +div {<br />
max-height:1000px;<br />
overflow:auto;<br />
margin:5px;<br />
}<br />
<br />
<br />
p{font-size:12px;<br />
font-color: #808080;<br />
}<br />
<br />
a{color:#006400;}<br />
<br />
<br />
</style><br />
</head><br />
<br />
<br />
<body><br />
<div class="textSpace"><br />
<br />
<br />
<div class="section"><br />
<br />
<div class="article"><br />
<h3 id="c1"><a href="#c1">week1(8/18/2013~8/25/2013)</a></h3><br />
<div><br />
<p><br />
<b>8/22//2013</b><br><br />
.Extracting the human ferritin plasmid and iron chaperone plasmid<br><br />
<b>8/23/2013</b><br><br />
.BBa_I746908 and BBa_I746902 transformation<br><br />
<b>8/24/2013</b><br><br />
.Extracting BBa_I746908 and BBa_I746902<br><br />
<b>8/25/2013</b><br><br />
<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="article"><br />
<h3 id="c2"><a href="#c2">week2(8/26/2013~9/1/2013)</a></h3><br />
<div><br />
<p><br />
<b>8/26/2013</b><br><br />
<br />
<b>8/27/2013</b><br><br />
<br />
<b>8/28/2013</b><br><br />
<br />
<b>8/29/2013</b><br><br />
.Gel electrophoresis(ferritin,iron chaperone4-6c,4-14I)<br><br />
.PCR:(4-6c),ferritin(mid,strong)<br><br />
.Digestion test:4-6c,ferritin,iron chaperone<br><br />
.Pre-culture:CRISPRi<br><br />
<br />
<br />
<b>8/30/2013</b><br><br />
.Extracting CRISPRi plasmids(fur,fieF,gor,and trxB)<br><br />
.PCR:ferritin(min,strong),CRISPRi(fur,fieF,for,and trxB)<br><br />
.Gel electrophresis:PCR products<br><br />
.Gel extractions:PCR products<br><br />
<b>8/31/2013</b><br><br />
.Digestion:ferritin(mid and strong),4-6c<br><br />
<br />
<b>9/1/2013</b><br><br />
.FASTR:CRISPRi<br><br />
.Ligation:ferritin and 4-6c<br><br />
.Begining editing Wiki<br><br />
<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="article"><br />
<h3 id="c3"><a href="#c3">week3(9/2/2013~9/7/2013)</a></h3><br />
<div><br />
<p><br />
<b>9/2/2013</b><br><br />
.Making plates with Broth and methylene blue(MB)<br><br />
<b>9/3/2013</b><br><br />
.Making olates with Broth and MB<br><br />
.Culturing BL21 and shuffle , respectively<br><br />
<b>9/4/2013</b><br><br />
<br />
<b>9/5/2013</b><br><br />
<br />
<b>9/6/2013</b><br><br />
<br />
<b>9/7/2013</b><br><br />
<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="article"><br />
<h3 id="c4"><a href="#c4">week4(9/8/2013~9/14/2013)</a></h3><br />
<div><br />
<p><br />
<br />
<b>9/9/2013</b><br><br />
.Hold a presentation(state of progress)<br><br />
.<br><br />
<b>9/10/2013</b><br><br />
<br />
<b>9/11/2013</b><br><br />
<br />
<b>9/12/2013</b><br><br />
<br />
<b>9/13/2013</b><br><br />
<br />
<b>9/14/2013</b><br><br />
<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br />
<br />
<h2 style="background-color:#ff9933"><center>Project</center></h2><br />
<br />
<center><br />
<table border="1"> <br />
<br />
<br />
<tr><br />
<br />
<td align="center"><br />
<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><br />
</td><br />
<br />
<br />
<td align="center"><br />
<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><br />
</td><br />
<br />
<td align="center"><br />
<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><br />
</td><br />
<br />
<br />
<br />
</tr><br />
<br />
<tr><br />
<br />
<th><br />
<a href="https://2013.igem.org/Team:Chiba/Project/uptake">Iron Uptake</a><br />
</th><br />
<br />
<th><br />
<a href="https://2013.igem.org/Team:Chiba/Project/store">Iron Storage</a><br />
</th><br />
<br />
<th><br />
<a href="https://2013.igem.org/Team:Chiba/Project/oxidation">Iron Oxidation</a><br />
</th><br />
<br />
</tr><br />
<br />
<br />
<br />
<br />
</table><br />
</center><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</div><br />
</body><br />
</html></div>T.Sendahttp://2013.igem.org/File:Chiba_SDS_no3s.pngFile:Chiba SDS no3s.png2013-09-27T20:10:54Z<p>T.Senda: </p>
<hr />
<div></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/storeTeam:Chiba/Project/store2013-09-27T19:59:54Z<p>T.Senda: </p>
<hr />
<div>{{Chiba_base}}<br />
<br />
<html xmlns="http://www.w3.org/1999/xhtml"><br />
<head><br />
<meta http-equiv="Content-Type" content="text/html;charset=utf-8"/><br />
<title>iGEM-2013 Chiba</title><br />
<br />
<style type="text/css"><br />
<br />
<br />
<br />
</style><br />
<br />
</head><br />
<br />
<body><br />
<div class="textSpace"><br />
<br />
<h2 id="store" style="background-color:#ff9933"><center>Storage</center></h2><br />
<br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<br />
<br><br />
<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp; In order to magnetize <i>E. coli</i>, we decided to import as much Fe ions as possible in <i>E. coli</i>. So, we focused on ferritin that stored Fe irons. Ferritin forms 24-mer protein composed by heavy chain (FTH) and light chain (FTL) as shown in Fig. 1. The H chain oxidizes Fe and L chain stores it. The reaction in H chain is below.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2Fe<sub>2</sub>+O<sub>2</sub>+(H<sub>2</sub>O)x+3→Fe<sub>2</sub>O<sub>2</sub>(H<sub>2</sub>O)x+4H+H<sub>2</sub>O<sub>2</sub><br><br />
The different species has the different ratio of FTH to FTL. Generally, the mammalian ferritin has more FTL on the other hand the ferritin derived from bacteria has more FTH. Because we wanted <i>E. coli</i> to store as much Fe as possible, the mammal ferritin is suitable for our object. Therefore, we decided to introduce human ferritin that can be expressed in <i>E.coli</i>.<br><br />
</br><br />
</br></br><br />
&nbsp;&nbsp;&nbsp;&nbsp;したがって、フェリチンの過剰発現によって、<br />
細胞の鉄の含有量は増加するはずである。また,実効的な2価鉄の濃度が減少し、細胞死が起こりにくくなるはずである。<br />
</p><br />
</br></br></br></br></br></br><br />
<br />
<br><br />
<p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ferrittin.png"alt=""align="middle"></center><br />
<center>Fig. 1. structure of ferritin<br></center><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Fe(II) in <i>E.coli</i> causes Fenton reaction in response to hydrogen peroxide and produce hydroxyl radical (OH・) which is harmful to <i>E.coli</i>. As a result, giving iron to <i>E.coli</i> excessively leads to the death of <i>E.coli</i>.<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;Ferritin is an intracellular protein which has a property to store iron. Ferritin has Heavy chain and Light chain. Heavy chain affects the oxidation of iron and stimulate 2Fe(II)+O<sub>2</sub>→[Fe(III)-O-O-Fe(III)] reaction. Light chain takes in Fe(III) in ferritin. Fe<sub>2</sub>O<sub>3</sub>(H<sub>2</sub>O) has paramagnetism.<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;These two effects enable isolation of iron in ferritin and enhance <i>E.coli</i> iron tolerance.<br />
<br />
</p><br />
</p><br />
<br />
<br />
<h3 style="background-color:#ffdead ">2.Materials & Methods</h3><br />
<br />
<p><br />
<h3 style="background-color:#f0ffff ">2.1.Parts</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp; The ferritin has FTH and FTL but the ratio is different in the species. However, it may be depend on the expression. So, we constructed two different human ferritin; BBa_K1057002 with middle RBS and BBa_K1057009 with strong RBS. Further more, we placed two ferritin genes (FTH and FTL) under pBAD promoter to control the expression level ant the timing. For these reasons, we constructed a new BioBrick based on BBa_I74608 (deposited by iGEM 2007 team Cambridge). <br><br />
<br><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/3/32/Chiba.ferritin.png"alt=""align="middle"></center><br />
<br />
<br />
<center><p>Fig. 2. Cloning of ferritin</p></center><br><br />
<br><br />
</p><br />
<br />
<br />
<a href="https://2013.igem.org/Team:Chiba/Parts"> Parts link</a><br />
</p><br />
<br />
<br />
<h3 style="background-color:#f0ffff ">2.2.Expression check</h3><br />
<p><br />
pBAD/araC-ferritin-strong and pBAD/araC-ferritin-mid were expressed individually in BL21 strain.<br><br />
We performed SDS-PAGE to check the expression of pBAD/araC-ferritin-strong and pBAD/araC-ferritin-mid.<br><br />
As a control, we conducted the same experiment with “sfgfp generator”.<br><br />
The results of SDS-PAGE were shown below.<br />
<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/8/8d/Chiba_SDS_no2.png" width="750px"height="500px"></center><br><br />
<center>Fig. 3:SDS-PAGE patterns of proteins extracted from <i>E. coli</i> introduced pBAD/araC-ferritin-strong, pBAD/araC-ferritin-mid, or BBa_I746908(sfgfp): (1)pBAD/araC-ferritin-strong, (2)pBAD/araC-ferritin-mid, (3)BBa_I746908(sfgfp)</center>. Left is the result of all fraction, and right is soluble fraction. There exist 2 bands around 20 kDa in both pBAD/araC-ferritin-strong and pBAD/araC-ferritin-mid. The band of 20 kDa is FTH. Another band, the band of 19 kDa, is FTL. There does not exist such bands in Lane(3), but exist a different band in 30 kDa. This is a band of sfgfp.<br />
<br />
<br />
<br />
</p><br />
<br />
<h3 style="background-color:#f0ffff ">2.3.鉄耐性の評価</h3><br />
<p><br />
<a href="https://2013.igem.org/Team:Chiba/Assay/store">Assay</a><br><br />
Experiment: We constructed two kinds of plasmids with which RBS scores are different as shown in Fig. 2. E. coli strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with above plasmids. Human ferritin genes (fth1 and ftl) are placed on the high-copy plasmid under the control of BAD promoter. To express Human ferritin proteins, arabinose was added into media(final conc. 0.2%). The resultant “ferritin generators” were cultured in the presence of iron citrate (Fe3+) or iron ascorbate (Fe2+), and checked final cell density and colony forming efficiency. As a control, we conducted the same experiment with “sfgfp generator”. <br />
</p><br />
<br />
<h3 style="background-color:#f0ffff ">2.4.Evaluation of magnetism</h3><br />
<p><br />
We examine whether BL21 overexpressing ferritin is attracted to a magnet.<br><br />
Our experimental setup is shown below(Fig. 3).<br><br />
<br />
The details are shown in <a href="https://2013.igem.org/Team:Chiba/Assay/store">assay.</a><br><br />
If <i>E. coli</i> have magnetism, the attract to magnets!<br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/e/e5/Chiba_magnet_method_ssp.png" width="600px"height="400px"></center><br><br />
<br><br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid</h3><br />
<br />
<h3 style="background-color:#f0ffff ">3.2 Assessment of iron tolerance</h3><br />
<br><p><b>Result. 1</b><br />
<br><left><img src="https://static.igem.org/mediawiki/2013/7/7f/Strorage_BL21_asc_OD_cor.PNG" width="377px"height="289px"></left><br />
<right><img src="https://static.igem.org/mediawiki/2013/f/f5/Storage_SHuffle_asc_OD_cor.PNG" width="372px"height="288px"></right><br><br />
<br><center>fig.A</center><br />
<br><br />
<br>Fig. A shows the optical density of <i>E. coli</i> which is cultured in the presence of iron for 12h (at 37°C).<br><br />
(Approximately 10^7 cells inoculated in to fresh media(2 mL, containing iron)in each.)<br />
<br>・Overexpression of ferritin didn’t affect the growth so much.<br />
<br>・The one without overexpression of ferritin, growth inhibition was observed when the concentration of ferrous ascorbate is 6-8mM. And when the concentration of ferrous ascorbate was over 8 mM, the growth of <i>E. coli</i> wasn’t observed at all.<br />
<br>・一方,フェリチンを過剰発現した大腸菌は,アスコルビン酸鉄濃度5-7 mMの範囲では,アスコルビン酸鉄不在下よりも増殖がよく?~ちょっとここ考えます,また9-10 mMのアスコルビン酸鉄濃度においても,細胞は増殖した<br />
<br>・<i>E. coli</i> strain <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> showed same results.<br />
<br />
<br />
<br />
<br><br><b>Result. 2</b><br />
<br><center><img src="https://static.igem.org/mediawiki/2013/3/39/Storage_result_BL_asc.PNG" width="716px"height="299px"></center><br><br />
<br>The ability to form colony changed similarly.<br />
<br><br />
<br />
<h3 style="background-color:#f0ffff ">3.3磁性評価</h3><br />
<p><b>Result</b><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/2a/Chiba_tetsu_BL21_str_no2.png" width="571px"height="500px"></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/1/10/Chiba_tetsu_BL21_mid_no2.png" width="571px"height="500px"></center><br><br />
Each <i>E. coli</i> don`t move at all.<br><br />
We consider this cause is uptake quantity of Fe(Ⅲ) is not sufficiently.<br><br />
So, expression levels of ferritin in each <i>E. coli</i> is not sufficiently.<br><br />
Future subject is to increase uptake quantity of Fe(Ⅲ) to knock down/out <i>Fur</i> and <i>fie</i>F.<br><br />
<br />
<br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">4.Conclusion</h3><br />
<br />
<p><br />
それは・・<br />
<br />
</p><br />
<br />
<br />
<br />
</div><br />
</body><br />
<br />
</html></div>T.Sendahttp://2013.igem.org/File:Chiba_SDS_no2.pngFile:Chiba SDS no2.png2013-09-27T19:57:21Z<p>T.Senda: </p>
<hr />
<div></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/storeTeam:Chiba/Project/store2013-09-27T19:55:46Z<p>T.Senda: </p>
<hr />
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<h2 id="store" style="background-color:#ff9933"><center>Storage</center></h2><br />
<br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<br />
<br><br />
<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp; In order to magnetize <i>E. coli</i>, we decided to import as much Fe ions as possible in <i>E. coli</i>. So, we focused on ferritin that stored Fe irons. Ferritin forms 24-mer protein composed by heavy chain (FTH) and light chain (FTL) as shown in Fig. 1. The H chain oxidizes Fe and L chain stores it. The reaction in H chain is below.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2Fe<sub>2</sub>+O<sub>2</sub>+(H<sub>2</sub>O)x+3→Fe<sub>2</sub>O<sub>2</sub>(H<sub>2</sub>O)x+4H+H<sub>2</sub>O<sub>2</sub><br><br />
The different species has the different ratio of FTH to FTL. Generally, the mammalian ferritin has more FTL on the other hand the ferritin derived from bacteria has more FTH. Because we wanted <i>E. coli</i> to store as much Fe as possible, the mammal ferritin is suitable for our object. Therefore, we decided to introduce human ferritin that can be expressed in <i>E.coli</i>.<br><br />
</br><br />
</br></br><br />
&nbsp;&nbsp;&nbsp;&nbsp;したがって、フェリチンの過剰発現によって、<br />
細胞の鉄の含有量は増加するはずである。また,実効的な2価鉄の濃度が減少し、細胞死が起こりにくくなるはずである。<br />
</p><br />
</br></br></br></br></br></br><br />
<br />
<br><br />
<p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ferrittin.png"alt=""align="middle"></center><br />
<center>Fig. 1. structure of ferritin<br></center><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Fe(II) in <i>E.coli</i> causes Fenton reaction in response to hydrogen peroxide and produce hydroxyl radical (OH・) which is harmful to <i>E.coli</i>. As a result, giving iron to <i>E.coli</i> excessively leads to the death of <i>E.coli</i>.<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;Ferritin is an intracellular protein which has a property to store iron. Ferritin has Heavy chain and Light chain. Heavy chain affects the oxidation of iron and stimulate 2Fe(II)+O<sub>2</sub>→[Fe(III)-O-O-Fe(III)] reaction. Light chain takes in Fe(III) in ferritin. Fe<sub>2</sub>O<sub>3</sub>(H<sub>2</sub>O) has paramagnetism.<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;These two effects enable isolation of iron in ferritin and enhance <i>E.coli</i> iron tolerance.<br />
<br />
</p><br />
</p><br />
<br />
<br />
<h3 style="background-color:#ffdead ">2.Materials & Methods</h3><br />
<br />
<p><br />
<h3 style="background-color:#f0ffff ">2.1.Parts</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp; The ferritin has FTH and FTL but the ratio is different in the species. However, it may be depend on the expression. So, we constructed two different human ferritin; BBa_K1057002 with middle RBS and BBa_K1057009 with strong RBS. Further more, we placed two ferritin genes (FTH and FTL) under pBAD promoter to control the expression level ant the timing. For these reasons, we constructed a new BioBrick based on BBa_I74608 (deposited by iGEM 2007 team Cambridge). <br><br />
<br><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/3/32/Chiba.ferritin.png"alt=""align="middle"></center><br />
<br />
<br />
<center><p>Fig. 2. Cloning of ferritin</p></center><br><br />
<br><br />
</p><br />
<br />
<br />
<a href="https://2013.igem.org/Team:Chiba/Parts"> Parts link</a><br />
</p><br />
<br />
<br />
<h3 style="background-color:#f0ffff ">2.2.Expression check</h3><br />
<p><br />
BL21株によってstr, midをそれぞれ発現させた。これによってフェリチンが発現されたかを確認するため、SDS-PAGEにより評価を行った。<br />
比較対象としてstrまたはmidの代わりにsfgfpをいれたプラスミドを発現したBL21株を用いた。<br />
以下に全画分および可溶性画分のSDS-PAGEを示す。<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/8/80/Chiba_SDS_sp2.png" width="750px"height="500px"></center><br><br />
<center>Fig. 3:SDS-PAGE patterns of proteins extracted from <i>E. coli</i> introduced pBAD/araC-ferritin-strong, pBAD/araC-ferritin-mid, or BBa_I746908(sfgfp): (1)pBAD/araC-ferritin-strong, (2)pBAD/araC-ferritin-mid, (3)BBa_I746908(sfgfp)</center>. Left is the result of all fraction, and right is soluble fraction. There exist 2 bands around 20 kDa in both pBAD/araC-ferritin-strong and pBAD/araC-ferritin-mid. The band of 20 kDa is FTH. Another band, the band of 19 kDa, is FTL. There does not exist such bands in Lane(3), but exist a different band in 30 kDa. This is a band of sfgfp.<br />
<br />
<br />
<br />
</p><br />
<br />
<h3 style="background-color:#f0ffff ">2.3.鉄耐性の評価</h3><br />
<p><br />
<a href="https://2013.igem.org/Team:Chiba/Assay/store">Assay</a><br><br />
Experiment: We constructed two kinds of plasmids with which RBS scores are different as shown in Fig. 2. E. coli strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with above plasmids. Human ferritin genes (fth1 and ftl) are placed on the high-copy plasmid under the control of BAD promoter. To express Human ferritin proteins, arabinose was added into media(final conc. 0.2%). The resultant “ferritin generators” were cultured in the presence of iron citrate (Fe3+) or iron ascorbate (Fe2+), and checked final cell density and colony forming efficiency. As a control, we conducted the same experiment with “sfgfp generator”. <br />
</p><br />
<br />
<h3 style="background-color:#f0ffff ">2.4.Evaluation of magnetism</h3><br />
<p><br />
We examine whether BL21 overexpressing ferritin is attracted to a magnet.<br><br />
Our experimental setup is shown below(Fig. 3).<br><br />
<br />
The details are shown in <a href="https://2013.igem.org/Team:Chiba/Assay/store">assay.</a><br><br />
If <i>E. coli</i> have magnetism, the attract to magnets!<br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/e/e5/Chiba_magnet_method_ssp.png" width="600px"height="400px"></center><br><br />
<br><br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid</h3><br />
<br />
<h3 style="background-color:#f0ffff ">3.2 Assessment of iron tolerance</h3><br />
<br><p><b>Result. 1</b><br />
<br><left><img src="https://static.igem.org/mediawiki/2013/7/7f/Strorage_BL21_asc_OD_cor.PNG" width="377px"height="289px"></left><br />
<right><img src="https://static.igem.org/mediawiki/2013/f/f5/Storage_SHuffle_asc_OD_cor.PNG" width="372px"height="288px"></right><br><br />
<br><center>fig.A</center><br />
<br><br />
<br>Fig. A shows the optical density of <i>E. coli</i> which is cultured in the presence of iron for 12h (at 37°C).<br><br />
(Approximately 10^7 cells inoculated in to fresh media(2 mL, containing iron)in each.)<br />
<br>・Overexpression of ferritin didn’t affect the growth so much.<br />
<br>・The one without overexpression of ferritin, growth inhibition was observed when the concentration of ferrous ascorbate is 6-8mM. And when the concentration of ferrous ascorbate was over 8 mM, the growth of <i>E. coli</i> wasn’t observed at all.<br />
<br>・一方,フェリチンを過剰発現した大腸菌は,アスコルビン酸鉄濃度5-7 mMの範囲では,アスコルビン酸鉄不在下よりも増殖がよく?~ちょっとここ考えます,また9-10 mMのアスコルビン酸鉄濃度においても,細胞は増殖した<br />
<br>・<i>E. coli</i> strain <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> showed same results.<br />
<br />
<br />
<br />
<br><br><b>Result. 2</b><br />
<br><center><img src="https://static.igem.org/mediawiki/2013/3/39/Storage_result_BL_asc.PNG" width="716px"height="299px"></center><br><br />
<br>The ability to form colony changed similarly.<br />
<br><br />
<br />
<h3 style="background-color:#f0ffff ">3.3磁性評価</h3><br />
<p><b>Result</b><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/2a/Chiba_tetsu_BL21_str_no2.png" width="571px"height="500px"></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/1/10/Chiba_tetsu_BL21_mid_no2.png" width="571px"height="500px"></center><br><br />
Each <i>E. coli</i> don`t move at all.<br><br />
We consider this cause is uptake quantity of Fe(Ⅲ) is not sufficiently.<br><br />
So, expression levels of ferritin in each <i>E. coli</i> is not sufficiently.<br><br />
Future subject is to increase uptake quantity of Fe(Ⅲ) to knock down/out <i>Fur</i> and <i>fie</i>F.<br><br />
<br />
<br />
</p><br />
<br />
<h3 style="background-color:#ffdead ">4.Conclusion</h3><br />
<br />
<p><br />
それは・・<br />
<br />
</p><br />
<br />
<br />
<br />
</div><br />
</body><br />
<br />
</html></div>T.Sendahttp://2013.igem.org/Team:Chiba/Project/storeTeam:Chiba/Project/store2013-09-27T19:54:44Z<p>T.Senda: </p>
<hr />
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<br />
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<h2 id="store" style="background-color:#ff9933"><center>Storage</center></h2><br />
<br />
<h3 style="background-color:#ffdead ">1.Introduction</h3><br />
<br />
<br><br />
<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp; In order to magnetize <i>E. coli</i>, we decided to import as much Fe ions as possible in <i>E. coli</i>. So, we focused on ferritin that stored Fe irons. Ferritin forms 24-mer protein composed by heavy chain (FTH) and light chain (FTL) as shown in Fig. 1. The H chain oxidizes Fe and L chain stores it. The reaction in H chain is below.<br><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2Fe<sub>2</sub>+O<sub>2</sub>+(H<sub>2</sub>O)x+3→Fe<sub>2</sub>O<sub>2</sub>(H<sub>2</sub>O)x+4H+H<sub>2</sub>O<sub>2</sub><br><br />
The different species has the different ratio of FTH to FTL. Generally, the mammalian ferritin has more FTL on the other hand the ferritin derived from bacteria has more FTH. Because we wanted <i>E. coli</i> to store as much Fe as possible, the mammal ferritin is suitable for our object. Therefore, we decided to introduce human ferritin that can be expressed in <i>E.coli</i>.<br><br />
</br><br />
</br></br><br />
&nbsp;&nbsp;&nbsp;&nbsp;したがって、フェリチンの過剰発現によって、<br />
細胞の鉄の含有量は増加するはずである。また,実効的な2価鉄の濃度が減少し、細胞死が起こりにくくなるはずである。<br />
</p><br />
</br></br></br></br></br></br><br />
<br />
<br><br />
<p><br />
<br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ferrittin.png"alt=""align="middle"></center><br />
<center>Fig. 1. structure of ferritin<br></center><br />
<p>&nbsp;&nbsp;&nbsp;&nbsp;Fe(II) in <i>E.coli</i> causes Fenton reaction in response to hydrogen peroxide and produce hydroxyl radical (OH・) which is harmful to <i>E.coli</i>. As a result, giving iron to <i>E.coli</i> excessively leads to the death of <i>E.coli</i>.<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;Ferritin is an intracellular protein which has a property to store iron. Ferritin has Heavy chain and Light chain. Heavy chain affects the oxidation of iron and stimulate 2Fe(II)+O<sub>2</sub>→[Fe(III)-O-O-Fe(III)] reaction. Light chain takes in Fe(III) in ferritin. Fe<sub>2</sub>O<sub>3</sub>(H<sub>2</sub>O) has paramagnetism.<br />
<br>&nbsp;&nbsp;&nbsp;&nbsp;These two effects enable isolation of iron in ferritin and enhance <i>E.coli</i> iron tolerance.<br />
<br />
</p><br />
</p><br />
<br />
<br />
<h3 style="background-color:#ffdead ">2.Materials & Methods</h3><br />
<br />
<p><br />
<h3 style="background-color:#f0ffff ">2.1.Parts</h3><br />
<p><br />
&nbsp;&nbsp;&nbsp;&nbsp; The ferritin has FTH and FTL but the ratio is different in the species. However, it may be depend on the expression. So, we constructed two different human ferritin; BBa_K1057002 with middle RBS and BBa_K1057009 with strong RBS. Further more, we placed two ferritin genes (FTH and FTL) under pBAD promoter to control the expression level ant the timing. For these reasons, we constructed a new BioBrick based on BBa_I74608 (deposited by iGEM 2007 team Cambridge). <br><br />
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<center><img src="https://static.igem.org/mediawiki/2013/3/32/Chiba.ferritin.png"alt=""align="middle"></center><br />
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<center><p>Fig. 2. Cloning of ferritin</p></center><br><br />
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<a href="https://2013.igem.org/Team:Chiba/Parts"> Parts link</a><br />
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<h3 style="background-color:#f0ffff ">2.2.Expression check</h3><br />
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BL21株によってstr, midをそれぞれ発現させた。これによってフェリチンが発現されたかを確認するため、SDS-PAGEにより評価を行った。<br />
比較対象としてstrまたはmidの代わりにsfgfpをいれたプラスミドを発現したBL21株を用いた。<br />
以下に全画分および可溶性画分のSDS-PAGEを示す。<br><br />
<center><img src="https://static.igem.org/mediawiki/2013/8/80/Chiba_SDS_sp2.png" width="750px"height="500px"></center><br><br />
<center>Fig. 3:SDS-PAGE patterns of proteins extracted from <i>E. coli</i> introduced pBAD/araC-ferritin-strong, pBAD/araC-ferritin-mid, or BBa_I746908(sfgfp): (1)pBAD/araC-ferritin-strong, (2)pBAD/araC-ferritin-mid, (3)BBa_I746908(sfgfp)</center>. Left is the result of all fraction, and right is soluble fraction. There exist 2 bands around 20 kDa in both pBAD/araC-ferritin-strong and pBAD/araC-ferritin-mid. The band of 20 kDa is FTH. Another band, the band of 19 kDa, is FTL. There does not exist such bands in Lane(3), but exist a different band in 30 kDa. This is a band of sfgfp.<br />
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<h3 style="background-color:#f0ffff ">2.3.鉄耐性の評価</h3><br />
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<a href="https://2013.igem.org/Team:Chiba/Assay/store">Assay</a><br><br />
Experiment: We constructed two kinds of plasmids with which RBS scores are different as shown in Fig. 2. E. coli strain BL21 and <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> were transformed with above plasmids. Human ferritin genes (fth1 and ftl) are placed on the high-copy plasmid under the control of BAD promoter. To express Human ferritin proteins, arabinose was added into media(final conc. 0.2%). The resultant “ferritin generators” were cultured in the presence of iron citrate (Fe3+) or iron ascorbate (Fe2+), and checked final cell density and colony forming efficiency. As a control, we conducted the same experiment with “sfgfp generator”. <br />
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<h3 style="background-color:#f0ffff ">2.4.Evaluation of magnetism</h3><br />
<p><br />
We examine whether BL21 overexpressing ferritin is attracted to a magnet.<br><br />
Our experimental setup is shown below(Fig. 3).<br><br />
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The details are shown in <a href="https://2013.igem.org/Team:Chiba/Assay/store">assay.</a><br><br />
If <i>E. coli</i> have magnetism, the attract to magnets!<br><br />
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<center><img src="https://static.igem.org/mediawiki/2013/e/e5/Chiba_magnet_method_ssp.png" width="600px"height="400px"></center><br><br />
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<h3 style="background-color:#ffdead ">3.Results & Discussion</h3><br />
<h3 style="background-color:#f0ffff ">3.1.plasmid</h3><br />
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<h3 style="background-color:#f0ffff ">3.2 Assessment of iron tolerance</h3><br />
<br><p><b>Result. 1</b><br />
<br><left><img src="https://static.igem.org/mediawiki/2013/7/7f/Strorage_BL21_asc_OD_cor.PNG" width="377px"height="289px"></left><br />
<right><img src="https://static.igem.org/mediawiki/2013/f/f5/Storage_SHuffle_asc_OD_cor.PNG" width="372px"height="288px"></right><br><br />
<br><center>fig.A</center><br />
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<br>Fig. A shows the optical density of <i>E. coli</i> which is cultured in the presence of iron for 12h (at 37°C).<br><br />
(Approximately 10^7 cells inoculated in to fresh media(2 mL, containing iron)in each.)<br />
<br>・Overexpression of ferritin didn’t affect the growth so much.<br />
<br>・The one without overexpression of ferritin, growth inhibition was observed when the concentration of ferrous ascorbate is 6-8mM. And when the concentration of ferrous ascorbate was over 8 mM, the growth of <i>E. coli</i> wasn’t observed at all.<br />
<br>・一方,フェリチンを過剰発現した大腸菌は,アスコルビン酸鉄濃度5-7 mMの範囲では,アスコルビン酸鉄不在下よりも増殖がよく?~ちょっとここ考えます,また9-10 mMのアスコルビン酸鉄濃度においても,細胞は増殖した<br />
<br>・<i>E. coli</i> strain <a href="http://www.nebj.jp/products/detail/113">SHuffle®</a> showed same results.<br />
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<br><br><b>Result. 2</b><br />
<br><center><img src="https://static.igem.org/mediawiki/2013/3/39/Storage_result_BL_asc.PNG" width="716px"height="299px"></center><br><br />
<br>The ability to form colony changed similarly.<br />
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<h3 style="background-color:#f0ffff ">3.3磁性評価</h3><br />
<p><b>Result</b<br />
<center><img src="https://static.igem.org/mediawiki/2013/2/2a/Chiba_tetsu_BL21_str_no2.png" width="571px"height="500px"></center><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/1/10/Chiba_tetsu_BL21_mid_no2.png" width="571px"height="500px"></center><br><br />
Each <i>E. coli</i> don`t move at all.<br><br />
We consider this cause is uptake quantity of Fe(Ⅲ) is not sufficiently.<br><br />
So, expression levels of ferritin in each <i>E. coli</i> is not sufficiently.<br><br />
Future subject is to increase uptake quantity of Fe(Ⅲ) to knock down/out <i>Fur</i> and <i>fie</i>F.<br><br />
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<h3 style="background-color:#ffdead ">4.Conclusion</h3><br />
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それは・・<br />
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</html></div>T.Senda