Team:Chiba/Project/store

From 2013.igem.org

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<h2 id="store" style="background-color:#ff9933"><center>Storage</center></h2>
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<h2 id="storage" style="background-color:#ff9933"><center>Sequestration: Fe-storage machine</center></h2>
<h3 style="background-color:#ffdead ">1.Introduction</h3>
<h3 style="background-color:#ffdead ">1.Introduction</h3>
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<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>.
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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>&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>
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<br>&nbsp;&nbsp;&nbsp;&nbsp;したがって、フェリチンの過剰発現によって、
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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>
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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。
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<br>
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<br>&nbsp;&nbsp;&nbsp;&nbsp;These two effects enable isolation of iron in ferritin and enhance <i>E.coli</i> iron tolerance.</p>
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<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ferrittin.png"alt=""align="middle"></center>
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</br></br></br></br></br></br></br></br></br>
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<center><p><b>Fig. 1</b> Complex structure of ferritin</p><br></center>
 +
<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>
 +
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>&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.
 +
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>
 +
<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>
 +
<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>
<p>
<p>
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&nbsp;&nbsp;&nbsp;&nbsp; In order to magnetize E. coli, we decided to import as much Fe ions as possible in E.coli. 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 Figure 1. The H chain oxidizes Fe and L chain stores it. The reaction in H chain is below.<br>
 
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2Fe2++O2+(H2O)x+3→Fe2O3(H2O)x+4H++H2O2<br>
 
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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 E. coli 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 E.coli.<br>
 
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<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ferrittin.png"alt=""align="middle"></center>
 
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<center>(Figure 1. structure of ferritin)<br></center>
 
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</p>
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<h3 style="background-color:#ffdead ">2.Experiments</h3>
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</p>
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<h3 style="background-color:#ffdead ">2.Materials!&!Methods</h3>
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<p>
<p>
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<h3 style="background-color:#f0ffff ">2.1.Parts</h3>
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<h3 style="background-color:#f0ffff ">2.1.BioBrick construction</h3>
<p>
<p>
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&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>
+
&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;  
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    <br><a href="http://parts.igem.org/Part:BBa_K1057002">BBa_K1057002</a>: 'middle' RBS assigned for FTH
 +
    <br><a href="http://parts.igem.org/Part:BBa_K1057009">BBa_K1057009</a>: 'strong' RBS assigned for FTH
 +
<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>
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<center><img src="https://static.igem.org/mediawiki/2013/3/3f/Chiba_ferittin.bio.png"alt=""align="middle"></center>
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<center><img src="https://static.igem.org/mediawiki/2013/2/23/Chiba.ferritin.cloning.png"alt=""align="middle"></center>
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<center>(Figure 2. a; BBa_I74608, b; BBa_K1057002/BBa_K1057009)</center><br>
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<center><p><b>Fig. 2</b> Cloning procedure of ferritin-producing BioBrick</p></center><br>
<br>
<br>
</p>
</p>
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<a href="#"> Parts link</a>
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<a href="https://2013.igem.org/Team:Chiba/Parts"> Parts link</a>
</p>
</p>
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<h3 style="background-color:#f0ffff ">2.2.Expression check</h3>
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<h3 style="background-color:#f0ffff ">2.2. Confirmation of Ferritin Expression </h3>
<p>
<p>
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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。
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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”.
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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。
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<br>
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以下に全画分および可溶性画分のSDS page を示す。<br>
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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.
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<img src="https://static.igem.org/mediawiki/2013/3/3e/Chiba_sds.png" width="750px"height="500px"><br>
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全画分、可溶性画分ともに、strong and middleには19kDaと20kDaにバンドが見られる。これらはそれぞれフェリチンのFTHとFTLに対応している。
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<br>
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また、sfgfpの発現株には、30kDa付近にsfgfpのバンドが見られる。
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<center><img src="https://static.igem.org/mediawiki/2013/4/4f/Chiba_ike.png" width="750px"height="500px"></center><br>
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これによってstr、およびmidでフェリチンが発現されたことが分かる。
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<center><p><b>Fig. 3</b> Expression of ferritin in <i>E. coli</i> treated with arabinose. 
 +
<br> lane 1. pBAD/araC-ferritin-strong(<a href="http://parts.igem.org/Part:BBa_K1057009">BBa_K1057009</a>)
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<br>lane 2. pBAD/araC-ferritin-mid(<a href="http://parts.igem.org/Part:BBa_K1057002">BBa_K1057002</a>)
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<br>lane 3. <a href="http://parts.igem.org/Part:BBa_I746908">BBa_I746908</a>(sfgfp)
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</p></center>
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</p>
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<h3 style="background-color:#f0ffff ">2.3.鉄耐性の評価</h3>
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<h3 style="background-color:#f0ffff ">2.3.Evaluation of iron tolerance</h3>
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<a href="#">Assay</a>
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<p>
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<h3 style="background-color:#f0ffff ">2.4.磁性評価</h3>
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<a href="#">Assay</a>
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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 (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>
 +
</p>
 +
 
 +
<h3 style="background-color:#f0ffff ">2.4.Evaluation of magnetism</h3>
 +
<p>
 +
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>.
 +
<center><img src="https://static.igem.org/mediawiki/2013/e/e5/Chiba_magnet_method_ssp.png" width="600px"height="400px"></center><br>
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<center><p><b>Fig. 4</b> Experimental setup</p></center>
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 +
<br>
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</p>
<h3 style="background-color:#ffdead ">3.Results & Discussion</h3>
<h3 style="background-color:#ffdead ">3.Results & Discussion</h3>
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<h3 style="background-color:#f0ffff ">3.1.プラスミド</a>
 
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<h3 style="background-color:#f0ffff ">3.2鉄耐性の評価</a>
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<h3 style="background-color:#f0ffff ">3.1 Assessment of iron tolerance</h3>
<br><p><b>Result. 1</b>
<br><p><b>Result. 1</b>
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<br>鉄培養液中で12時間培養した後の大腸菌の光学密度を測定するとfig.Aになった。
 
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<br>(鉄培養液に植菌した際の細胞光学密度は約0.01 (OD600)に統一した)
 
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<br>
 
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<br>・フェリチンの過剰発現発現は増殖に大きな影響を与えなかった。
 
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<br>・フェリチンを過剰発現していないコントロールの場合,アスコルビン酸鉄濃度が6-8 mMのときに,明らかな増殖阻害がみられ,8 mM以上では,大腸菌の生育が全く見られなかった。
 
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<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>
 +
<center><img src="https://static.igem.org/mediawiki/2013/8/81/Chiba_last.jpg" ></center><br>
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<br><center><p><b>Fig. 5</b> Iron tolerance of <i>E. coli</i> harboring ferritin-expressing BioBricks</p></center>
<br>
<br>
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<br><b>Ressult. 2</b>
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<br></p><p>
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<br>コロニー形成能の変化も,ほぼ同様の傾向を示した<br><p>
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<br>•Over-expression of ferritin didn’t affect the growth so much. <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>
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<h3 style="background-color:#f0ffff ">3.3磁性評価</a>
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•The cell expressing human ferritins showed significantly higher tolerance to the ferrous ascorbate. <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>
 +
•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><center><img src="https://static.igem.org/mediawiki/2013/8/86/Chiba_ike_miracle.png" width="716px"></center><br>
 +
<br><center><p><b>Fig. 6</b>  Colony-forming capability of <i>E. coli</i> with/without ferritin-expressing BioBricks</p></center><br>
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<h3 style="background-color:#ffdead ">4.Conclusion</h3>
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<h3 style="background-color:#f0ffff ">3.2 Evaluation of magnetism</h3>
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<p><b>Result</b><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.
 +
Alas!, however,  we did not see any trends of E. coli flock forming around magnet: nothing really happened....
 +
<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>
 +
<center><img src="https://static.igem.org/mediawiki/2013/9/93/Chiba_tetsu_BL21_str_ssp.png" width="571px"></center><br>
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<center><img src="https://static.igem.org/mediawiki/2013/b/b2/Chiba_tetsu_BL21_mid_sp.png" width="571px"></center><br>
<p>
<p>
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それは・・
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<center><p><b>Fig. 7</b> Our first attempt to attract <i>E. coli</i> by magnet.</p></center>
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</p>
</p>

Latest revision as of 04:19, 28 September 2013

iGEM-2013 Chiba

iGEM-2013 Chiba

Sequestration: Fe-storage machine

1.Introduction


    

Fe must be isolated: In order to magnetize E. coli, we need to stuff as much Fe ions as possible in E. coli. 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.



Fe container machinery: 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.


Fig. 1 Complex structure of ferritin


    Heavy chain catalyzes oxidation of iron and stimulate 2Fe(II)+O2→[Fe(III)-O-O-Fe(III)] reaction

2Fe2+O2+(H2O)x+3→Fe2O2(H2O)x+4H+H2O2

    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. 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.

Choice of ferritin: Because the storage capacity of E. coli ferritin is far less than that of mammarian type, we decided to make BioBrick for the functional expression of human ferritin in E. coli.

Hypothesis: By storing Fe in isolation, the maximum capacity for Fe storage should be elevated. Also, the apparent iron tolerance of E. coli should be also elevated.


2.Experiments

2.1.BioBrick construction

    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;
BBa_K1057002: 'middle' RBS assigned for FTH
BBa_K1057009: 'strong' RBS assigned for FTH
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.

Fig. 2 Cloning procedure of ferritin-producing BioBrick



Parts link

2.2. Confirmation of Ferritin Expression

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”.
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.


Fig. 3 Expression of ferritin in E. coli treated with arabinose.
lane 1. pBAD/araC-ferritin-strong(BBa_K1057009)
lane 2. pBAD/araC-ferritin-mid(BBa_K1057002)
lane 3. BBa_I746908(sfgfp)

2.3.Evaluation of iron tolerance

Experiment: E. coli strain BL21 and SHuffle® 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 BBa_I746908 (arabinose-induced sfGFP generator). For the detail of the iron torelance protocol, see Here.

2.4.Evaluation of magnetism

Now, we finally examined whether BL21 overexpressing ferritin is well attracted to the magnet. Our experimental setup is shown below (Fig. 4).
The details of this experimental protocol is given here.


Fig. 4 Experimental setup


3.Results & Discussion

3.1 Assessment of iron tolerance


Result. 1



Fig. 5 Iron tolerance of E. coli harboring ferritin-expressing BioBricks




•Over-expression of ferritin didn’t affect the growth so much.
•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 SHuffle®) above that concentration.
•The cell expressing human ferritins showed significantly higher tolerance to the ferrous ascorbate.
•We observed significant difference in viability profiles of the two strains BL21 and SHuffle®. 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 SHuffle® 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 SHuffle® is specifically engineered strain so that the cytosolic environment is more oxidizing than the normal E. coli cells.
•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.



Fig. 6 Colony-forming capability of E. coli with/without ferritin-expressing BioBricks


3.2 Evaluation of magnetism

Result
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. Alas!, however, we did not see any trends of E. coli flock forming around magnet: nothing really happened....

Maybe, we should have done the same experiment in SHuffle® 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 Fur and fieF using CRISPRi technique.



Fig. 7 Our first attempt to attract E. coli by magnet.