Difference between revisions of "Team:TJU/Results"

 
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             </li>
 
             </li>
 
             <li class="hmain"><a href="https://2015.igem.org/Team:TJU/Results">Results</a>  
 
             <li class="hmain"><a href="https://2015.igem.org/Team:TJU/Results">Results</a>  
 +
            <ul>
 +
                    <li>
 +
                    <a href="https://2015.igem.org/Team:TJU/Results#Lactate producing system">Lactate producing system</a>
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                    </li>
 +
                    <li>
 +
                        <a href="https://2015.igem.org/Team:TJU/Results#Flavins producing system">Flavins producing system</a>
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                    </li>
 +
                    <li>
 +
                        <a href="https://2015.igem.org/Team:TJU/Results#Co-culture MFC">Co-culture MFC</a>
 +
                    </li>
 +
                </ul>
 +
 
             </li>  
 
             </li>  
 
             <li class="hmain"><a href="https://2015.igem.org/Team:TJU/Future Work">Future Work</a>  
 
             <li class="hmain"><a href="https://2015.igem.org/Team:TJU/Future Work">Future Work</a>  
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     <div id="body-PART-r" >
 
     <div id="body-PART-r" >
 
     </br>
 
     </br>
     <a name="Results" id="Results"></a><div align="center"><h2>Results</h2></div>
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     <div align="center"><h2>Results</h2></div>
 
     </br>
 
     </br>
<a name="1 Lactate producing system" id="1 Lactate producing system"></a><h3 >1 Lactate producing system</h3><hr>
 
1.    <div class="kuang" style="width:470px">
 
</br><a href="https://static.igem.org/mediawiki/2015/4/44/Result_1.png
 
" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/4/44/Result_1.png
 
"  width="450"  alt=""/></a>
 
<div id="Enlarge">         
 
<p> <b>Figure 1.</b> <span style="font-size: 14px"> 图要改  111</span>
 
<a href="https://static.igem.org/mediawiki/2015/4/44/Result_1.png
 
"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
 
<p>Under anaerobic conditions, wild-type MG1655 has the same growth rate as MG1655 with Δ<span style="font-style: italic">pflB</span> and MG1655 with  Δ<span style="font-style: italic">pflB</span> together with the insertion of <span style="font-style: italic">ldhE</span> keeps relatively lower growth rate than the two mentioned before.</p></br>
 
2. Glucose consumption curve
 
<p>The anaerobically consumption rate keeps the nearly same among MG1655, MG1655Δ<span style="font-style: italic">pflB</span> and MG1655Δ<span style="font-style: italic">pflB</span>+<span style="font-style: italic">ldhE</span>.</p></br>
 
3和4乳酸生成曲线 lactate production curve
 
<p>In anaerobic environment, lactate production of MG1655, MG1655 Δ<span style="font-style: italic">pflB</span> and MG1655 Δ<span style="font-style: italic">pflB</span>+<span style="font-style: italic">ldhE</span> has large differences between each other. Notably, knocking out of <span style="font-style: italic">pflB</span> will increase lactate generation up to ~2 g/L and the amount produced by MG1655Δ<span style="font-style: italic">pflB</span>+<span style="font-style: italic">ldhE</span> is slightly greater than MG1655Δ<span style="font-style: italic">pflB</span>.</p>
 
<p>Conclusion: knocking out of <span style="font-style: italic">pflB</span> can dramatically improve the lactate production. Besides, <span style="font-style: italic">ldhE</span> also plays an important role in lactate increase. We improve our utilization of carbon sources by constructing a high-yield strain with M9 medium under the same concentrations of glucose.</p>
 
  
 +
    <a name="Lactate producing system" id="Lactate producing system"></a><h3>1 Lactate producing system </h3> <hr></br>
 +
<p>In order to verify our <span style="font-style: italic">ldhE</span> part and <span style="font-style: italic">pflB</span> knockout strategy have works well, we get following results. </p></br>
  
<a name="Flavins producing system" id="Flavins producing system"></a><h3> 2 Flavins producing system </h3> <hr></br>
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<div class="kuang" style="width:770px">
 
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</br><a href="https://static.igem.org/mediawiki/2015/5/5e/Result_1%27.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/5/5e/Result_1%27.png"  width="750"  alt=""/></a>
<a name="Co-culture MFC" id="Co-culture MFC"></a><h3> 3 Co-culture MFC -- <span style="font-style: normal; color:#ddbf73;">Labor Division</span> </h3> <hr></br>
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<div id="Enlarge">        
 +
<p> <b>Figure 1.</b> <span style="font-size: 14px"> a: The growth curve in anaerobic condition (M9 medium).  b: Glucose consumption curve in anaerobic condition (M9 medium).</span>
 +
<a href="https://static.igem.org/mediawiki/2015/5/5e/Result_1%27.png"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
  
 +
<p>Shown as figure 1a, under anaerobic conditions, wild-type MG1655 has the same growth rate as MG1655Δ<span style="font-style: italic">pflB</span>, but MG1655Δ<span style="font-style: italic">pflB</span> + <span style="font-style: italic">ldhE</span> keeps relatively lower growth rate than the two strains mentioned before. Shown as figure 1b, the anaerobic glucose consumption rate keeps the nearly same among MG1655, MG1655Δ<span style="font-style: italic">pflB</span> and MG1655Δ<span style="font-style: italic">pflB</span> + <span style="font-style: italic">ldhE</span>.</p></br>
  
<div class="kuang" style="width:470px">
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<div class="kuang" style="width:770px">
</br><a href="https://static.igem.org/mediawiki/2015/3/3d/R-PART_2.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/3/3d/R-PART_2.png"  width="450"  alt=""/></a>
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</br><a href="https://static.igem.org/mediawiki/2015/1/16/Result_2%281%29.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/1/16/Result_2%281%29.png"  width="750"  alt=""/></a>
 
<div id="Enlarge">           
 
<div id="Enlarge">           
<p> <b>Figure 1.</b> <span style="font-size: 14px"> 图要改 111</span>
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<p> <b>Figure 2.</b> <span style="font-size: 14px"> a: The lactate production curve in anaerobic condition (M9 medium). b: The lactate production in anaerobic condition (M9 medium, 25h after inoculation)</span>
<a href="https://static.igem.org/mediawiki/2015/3/3d/R-PART_2.png"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
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<a href="https://static.igem.org/mediawiki/2015/1/16/Result_2%281%29.png"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
  
<div class="kuang" style="width:470px">
+
<p>In anaerobic environment, lactate production of MG1655, MG1655Δ<span style="font-style: italic">pflB</span> and MG1655Δ<span style="font-style: italic">pflB</span>+<span style="font-style: italic">ldhE</span> has large differences between each other. Notably, knocking out of <span style="font-style: italic">pflB</span> together with <span style="font-style: italic">ldhE</span> insertion will increase lactate generation up to ~3 g/L and the amount produced by MG1655Δ<span style="font-style: italic">pflB</span> is smaller than MG1655Δ<span style="font-style: italic">pflB</span>+<span style="font-style: italic">ldhE</span>.</p></br>
</br><a href="https://static.igem.org/mediawiki/2015/0/05/MFC-1.png
+
" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/0/05/MFC-1.png
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"  width="450" alt=""/></a>
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<div id="Enlarge">        
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<p> <b>Figure 1.</b> <span style="font-size: 14px"> 图要改  111</span>
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<a href="https://static.igem.org/mediawiki/2015/0/05/MFC-1.png
+
"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
+
  
<div class="kuang" style="width:470px">
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<p>So, knocking out of <span style="font-style: italic">pflB</span> can dramatically improve the lactate production. Besides, <span style="font-style: italic">ldhE</span> also plays an important role in lactate increase. Engineered lactate producing strain (MG1655Δ<span style="font-style: italic">pflB</span>+<span style="font-style: italic">ldhE</span>) has a greater utilization of carbon sources and a higher yield of lactate with M9 medium under the same concentrations of glucose.</p></br>
</br><a href="https://static.igem.org/mediawiki/2015/3/35/MFC-2.png
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" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/3/35/MFC-2.png
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"   width="450"  alt=""/></a>
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<div id="Enlarge">         
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<p> <b>Figure 1.</b> <span style="font-size: 14px"> 图要改  111</span>
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<a href="https://static.igem.org/mediawiki/2015/3/35/MFC-2.png
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"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
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<div class="kuang" style="width:470px">
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<a name="Flavins producing system" id="Flavins producing system"></a><h3> 2 Flavins producing system </h3> <hr></br>
</br><a href="https://static.igem.org/mediawiki/2015/a/ad/MFC-3.png
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" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/a/ad/MFC-3.png
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"  width="450"  alt=""/></a>
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<div id="Enlarge">        
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<p> <b>Figure 1.</b> <span style="font-size: 14px"> 图要改  111</span>
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<a href="https://static.igem.org/mediawiki/2015/a/ad/MFC-3.png
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"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
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<div class="kuang" style="width:470px">
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<p>To prove the EC10 and EC10* work as expected, we conduct several experiments and the results are as follows.</p></br>
</br><a href="https://static.igem.org/mediawiki/2015/8/8b/MFC-4.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/8/8b/MFC-4.png"  width="450"  alt=""/></a>
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<div id="Enlarge">         
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<p> <b>Figure 1.</b> <span style="font-size: 14px"> 图要改  111</span>
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<a href="https://static.igem.org/mediawiki/2015/8/8b/MFC-4.png"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
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<div class="kuang" style="width:470px">
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<div class="kuang" style="width:570px">
</br><a href="https://static.igem.org/mediawiki/2015/1/19/MFC-5.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/1/19/MFC-5.png"  width="450"  alt=""/></a>
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</br><a href="https://static.igem.org/mediawiki/2015/2/20/QQ%E5%9B%BE%E7%89%8720150918005538.jpg" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/2/20/QQ%E5%9B%BE%E7%89%8720150918005538.jpg"  width="550"  alt=""/></a>
 
<div id="Enlarge">           
 
<div id="Enlarge">           
<p> <b>Figure 1.</b> <span style="font-size: 14px"> 图要改  111</span>
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<p> <b>Figure 3.</b> <span style="font-size: 14px"> The yield of riboflavin in different strains: EC10, Rf02S (Δ<span style="font-style: italic">pgi</span> + EC10), EC10*</span>
<a href="https://static.igem.org/mediawiki/2015/1/19/MFC-5.png"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
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<a href="https://static.igem.org/mediawiki/2015/2/20/QQ%E5%9B%BE%E7%89%8720150918005538.jpg"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
  
<div class="kuang" style="width:470px">
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<p>As shown in figure 4, <span style="font-style: italic">E. coli</span> BL21 with EC10 reaches a final yield of 17 mg/L in flask cultivation. <span style="font-style: italic">E. coli</span> BL21Δ<span style="font-style: italic">pgi</span> + EC10 reaches 33 mg/L. <span style="font-style: italic">E. coli</span> BL21 + EC10* reaches 90 mg/L.</p></br>
</br><a href="https://static.igem.org/mediawiki/2015/e/e4/MFC-6.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/e/e4/MFC-6.png"   width="450"  alt=""/></a>
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<div id="Enlarge">         
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<p> <b>Figure 1.</b> <span style="font-size: 14px"> 图要改  111</span>
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<a href="https://static.igem.org/mediawiki/2015/e/e4/MFC-6.png"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
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<div class="kuang" style="width:470px">
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<div class="kuang" style="width:590px">
</br><a href="https://static.igem.org/mediawiki/2015/5/56/MFC-7.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/5/56/MFC-7.png"  width="450"  alt=""/></a>
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</br><a href="https://static.igem.org/mediawiki/2015/2/25/Ribo-4.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/2/25/Ribo-4.png"  width="570"  alt=""/></a>
 
<div id="Enlarge">           
 
<div id="Enlarge">           
<p> <b>Figure 1.</b> <span style="font-size: 14px"> 图要改  111</span>
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<p> <b>Figure 4.</b> <span style="font-size: 14px"> The yield of riboflavin in different strains :EC10, Rf02S (Δ<span style="font-style: italic">pgi</span>+EC10), EC10* </span>
<a href="https://static.igem.org/mediawiki/2015/5/56/MFC-7.png"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
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<a href="https://static.igem.org/mediawiki/2015/2/25/Ribo-4.png"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
  
<div class="kuang" style="width:470px">
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<a name="Co-culture MFC" id="Co-culture MFC"></a><h3> 3 Co-culture MFC -- <span style="font-style: normal; color:#ddbf73;">Division Of Labor</span> </h3> <hr></br>
</br><a href="https://static.igem.org/mediawiki/2015/6/60/MFC-8.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/6/60/MFC-8.png"   width="450"  alt=""/></a>
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<div id="Enlarge">         
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<p> <b>Figure 1.</b> <span style="font-size: 14px"> 图要改  111</span>
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<a href="https://static.igem.org/mediawiki/2015/6/60/MFC-8.png"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
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<div class="kuang" style="width:470px">
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<div class="kuang" style="width:770px">
</br><a href="https://static.igem.org/mediawiki/2015/f/f5/MFC-9.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/f/f5/MFC-9.png"  width="450"  alt=""/></a>
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</br><a href="https://static.igem.org/mediawiki/2015/c/c1/Result_5'.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/c/c1/Result_5'.png"  width="750"  alt=""/></a>
 
<div id="Enlarge">           
 
<div id="Enlarge">           
<p> <b>Figure 1.</b> <span style="font-size: 14px"> 图要改  111</span>
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<p> <b>Figure 5.</b> <span style="font-size: 14px"> The electrical output of MFCs of single strain <span style="font-style: italic">Shewanella oneidensis</span> MR-1 with different substrates.</span>
<a href="https://static.igem.org/mediawiki/2015/f/f5/MFC-9.png"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
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<a href="https://static.igem.org/mediawiki/2015/c/c1/Result_5'.png"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
  
<div class="kuang" style="width:470px">
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<p>From the diagram above, we demonstrate that:</p>
</br><a href="https://static.igem.org/mediawiki/2015/7/76/MFC-10.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/7/76/MFC-10.png"  width="450"  alt=""/></a>
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<p>  1. <span style="font-style: italic">Shewanella</span> cannot use glucose as a mere carbon substrate to generate power and so the potential keeps no more than 50 mV. </p>           
 +
<p>  2. <span style="font-style: italic">Shewanella</span> can produce the maximal potential up to 150mV with supplement of sodium lactate. </p>
 +
<p>  3. <span style="font-style: italic">Shewanella</span> can produce even higher potential when we take sodium lactate as the carbon source and add little riboflavin into the system.</p></br>
 +
<p>Conclusion: According to our experiment, our system is feasible in principle. Lactate can serve as the entry point in material flow and riboflavin as the major factor in energy and information flow, which lays a foundation in the later experiment design. We can use fermentation bacteria to provide carbon sources and electron shuttles, which subsequently, will increase the power generation capability in the co-culture system.</p></br>
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<div class="kuang" style="width:770px">
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</br><a href="https://static.igem.org/mediawiki/2015/f/f9/Result_6.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/f/f9/Result_6.png"  width="750"  alt=""/></a>
 
<div id="Enlarge">           
 
<div id="Enlarge">           
<p> <b>Figure 1.</b> <span style="font-size: 14px"> 图要改  111</span>
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<p> <b>Figure 6.</b> <span style="font-size: 14px"> The electrical output of co-culture MFCs with preliminary division of labor</span>
<a href="https://static.igem.org/mediawiki/2015/7/76/MFC-10.png"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
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<a href="https://static.igem.org/mediawiki/2015/f/f9/Result_6.png"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
  
<p>Figure 9: Among optimized <span style="font-style: italic">shewanella</span>, <span style="font-style: italic">shewanella</span>+MG1655 and <span style="font-style: italic">Shewanella</span>+Δ<span style="font-style: italic">plfB</span> <span style="font-style: italic">ldhE</span>+Rf02S, the three-strain coculture system reached a relatively higher potential that kept around 350 mV in 80 hours.</p></br>
+
<p>Description:
<p>Conclusion: <span style="font-style: italic">Shewanella</span>+Δ<span style="font-style: italic">plfB</span> <span style="font-style: italic">ldhE</span>+Rf02S has been going through a preferable optimization. It led to the potential of 300 mV in 80 hours under the condition of 2 g/L glucose without any supplementary. So, it realized a higher and more endurable power generation when taking glucose as carbon sources in <span style="font-style: italic">Shewanella</span>.</p></br>
+
The results show that dividing material, energy and information flow into separated fermentation bacteria is much better than combining the two tasks into one kind of bacteria. We suppose that the production of riboflavins and lactate cannot be consistent in single strain because lactate is the primary metabolites and riboflavin is the secondary.</p>
  
<p>Figure 10: The potential and time of duration in <span style="font-style: italic">Shewanella</span>+Δ<span style="font-style: italic">plfB</span> <span style="font-style: italic">ldhE</span>+<span style="font-style: italic">B.Subtilis</span> system can be raised into 511 mV and 80 h, respectively, resulted in a greater generation amount.</p></br>
+
<p>However, the electricity output cannot sustain for long and decrease promptly (as shown in figure 5).Based on the measurement, samples from anode reaches an excessively low pH (lower than 6.2) which might not meet the survival requirement of <span style="font-style: italic">Shewanella</span>.</p>
  
<p>Figure 11:</p>
+
<p>To optimize the system, we try to control the rate of lactate production and adjust the proportion of fermentation bacteria.</p></br>
<p>The potential produce by <span style="font-style: italic">Shewanella</span>+Δ<span style="font-style: italic">plfB</span> <span style="font-style: italic">ldhE</span>+<span style="font-style: italic">B.Subtilis</span> is greater than <span style="font-style: italic">Shewanella</span>+Δ<span style="font-style: italic">plfB</span> <span style="font-style: italic">ldhE</span>+Rf02S for 176 mV. Cocultured <span style="font-style: italic">Shewanella</span>, <span style="font-style: italic">E.coli</span> and <span style="font-style: italic">B.subtilis</span> can achieve a better job division that can reduce the competition between two kinds of zymophyte since <span style="font-style: italic">B,subtilis</span> can anaerobically metabolize by using KNO<sub>3</sub>. It significantly improve the commensalism relations between three species.</p></br>
+
  
 +
<div class="kuang" style="width:770px">
 +
</br><a href="https://static.igem.org/mediawiki/2015/0/01/Result_00.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/0/01/Result_00.png"  width="750"  alt=""/></a>
 +
<div id="Enlarge">         
 +
<p> <b>Figure 7.</b> <span style="font-size: 14px"> The electrical output of co-culture MFCs with optimized division of labor and bacteria proportion.</span>
 +
<a href="https://static.igem.org/mediawiki/2015/0/01/Result_00.png"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
  
<p>Figure 12</p>
+
<p>The maximal potential increased up to 322 mV and could maintain for 30 hours in the optimized three-strain system. (consist of <span style="font-style: italic">Shewanella</span> and two kinds of <span style="font-style: italic">E. coli</span>)</p></br>
(1) A series of voltage brought by different external resistance from <span style="font-style: italic">Shewanella</span><span style="font-style: italic">plfB</span> <span style="font-style: italic">ldhE</span>+Rf02S</p>
+
(2) The relations between current density and voltage is well suited to polarization curve while the power curve can represent the relations between output power density and current density. Besides, the highest power density can reach to 10 mW/m<sup>2</sup>.</p></br>
+
  
<p>Figure 13</p>
+
<div class="kuang" style="width:770px">
<p>(1) A series of voltage brought by different external resistance from <span style="font-style: italic">Shewanella</span><span style="font-style: italic">plfB</span> <span style="font-style: italic">ldhE</span>+<span style="font-style: italic">B.Subtilis</span></p>
+
</br><a href="https://static.igem.org/mediawiki/2015/3/3b/Result_8%27.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/3/3b/Result_8%27.png"   width="750"  alt=""/></a>
<p>(2) The relations between current density and voltage is well suited to polarization curve while the power curve can represent the relations between output power density and current density. Besides, the highest power density can reach to 17 mW/m<sup>2</sup>.</p></br>
+
<div id="Enlarge">        
 +
<p> <b>Figure 8.</b> <span style="font-size: 14px"> The electrical output of co-culture system with fermentation bacteria added 8h later into the system</span>
 +
<a href="https://static.igem.org/mediawiki/2015/3/3b/Result_8%27.png"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
  
  
<p>Figure 14</p>
+
<div class="kuang" style="width:770px">
The comparison of polarization curve and power curve among <span style="font-style: italic">shewanella</span>, <span style="font-style: italic">shewanella</span>+MG1655, <span style="font-style: italic">shewanella</span>+Δ<span style="font-style: italic">plfB</span> <span style="font-style: italic">ldhE</span>+Rf02S. It is obvious that the power output in <span style="font-style: italic">shewanella</span>+Δ<span style="font-style: italic">plfB</span> <span style="font-style: italic">ldhE</span>+Rf02S is far more bigger than the controled group.</p> </br> 
+
</br><a href="https://static.igem.org/mediawiki/2015/6/6d/Result_12%27.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/6/6d/Result_12%27.png"  width="750" alt=""/></a>
 
+
<div id="Enlarge">        
 
+
<p> <b>Figure 12.</b> <span style="font-size: 14px"> a
<p>Figure 15</p>
+
(1) A series of voltage brought by different external resistance from <span style="font-style: italic">Shewanella</span> + Δ<span style="font-style: italic">pflB</span> <span style="font-style: italic">ldhE</span> + Rf02S MFC system
<p>The comparison of polarization curve and power curve among <span style="font-style: italic">shewanella</span>, <span style="font-style: italic">shewanella</span>+MG1655 and <span style="font-style: italic">Shewanella</span>+Δ<span style="font-style: italic">plfB</span> <span style="font-style: italic">ldhE</span>+<span style="font-style: italic">B.Subtilis</span>. We can apparently observed that the power output of <span style="font-style: italic">Shewanella</span>+Δ<span style="font-style: italic">plfB</span> <span style="font-style: italic">ldhE</span>+<span style="font-style: italic">B.Subtilis</span> is greater than the controled group.</p></br>
+
(2)The polarization curve and power density of <span style="font-style: italic">Shewanella</span> + Δ<span style="font-style: italic">pflB</span> <span style="font-style: italic">ldhE</span> + Rf02S MFC system.  
 
+
Besides, the highest power density can reach to 10mW/m<sup>2</sup>.
<p>Figure 16</p>
+
b
<p>The comparison of polarization curve and power curve among <span style="font-style: italic">Shewanella</span>+Δ<span style="font-style: italic">plfB</span> <span style="font-style: italic">ldhE</span>+<span style="font-style: italic">B.Subtilis</span> and <span style="font-style: italic">Shewanella</span>+Δ<span style="font-style: italic">plfB</span> <span style="font-style: italic">ldhE</span>+Rf02S. It has been shown in power curve that <span style="font-style: italic">Shewanella</span>+Δ<span style="font-style: italic">plfB</span> <span style="font-style: italic">ldhE</span>+<span style="font-style: italic">B.Subtilis</span> has a higher output than <span style="font-style: italic">Shewanella</span>+Δ<span style="font-style: italic">plfB</span> <span style="font-style: italic">ldhE</span>+Rf02S, which indicates that three-strain system can generate higher electricity and have a better MFC performance, in return, have a promising application.</p></br>
+
(1)A series of voltage brought by different external resistance from <span style="font-style: italic">Shewanella</span> + Δ<span style="font-style: italic">pflB</span> <span style="font-style: italic">ldhE</span> + <span style="font-style: italic">B. subtilis</span>
   
+
(2)The polarization curve and power density of <span style="font-style: italic">Shewanella</span> + Δ<span style="font-style: italic">pflB</span> <span style="font-style: italic">ldhE</span> + <span style="font-style: italic">B. subtilis</span> MFC system.
    <div align="center"><h2>Future work</h2></div>
+
</span>
    </br>
+
<a href="https://static.igem.org/mediawiki/2015/6/6d/Result_12%27.png" target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
<a name="Proteolysis" id="Proteolysis"></a><h3> Proteolysis system </h3> <hr></br>
+
<p>During the experiment, our MFC in anaerobic condition has been found to reach an unexpected lower pH that showed harmful to bacterial consortium. On account of improving the rate of survival, we designed a “Sensing- Regulating” system to consume the accumulated lactate and keep the environment at a proper acid level. Our system belongs to proteomic regulation, which contains pH sensing system and orthogonal proteolysis system for self-regulating. The delayed effect within our sensing system brings us a more precise depict of possible result that the pH may oscillate at a certain range. </p></br>
+
  
<p style="font-weight: bolder">1  Sensing</p>
+
<p>The relations between current density and voltage is referred as polarization curve while the power curve can represent the relations between output power density and current density. We can learn from the figure 13, the highest power density of (1) reaches 10 mW/m<sup>2</sup> and (2) reaches 17 mW/m<sup>2</sup>.</p></br>
<p>Every genus of cell has an optimum range of pH to live and <span style="font-style: italic">Shewanella</span> along with <span style="font-style: italic">E.coli</span> is no exception. Previous research has identified that <span style="font-style: italic">Shewanella</span> can maintain a maximal population density when pH keeps above 6.2 while <span style="font-style: italic">E.coli</span> can survive in 5.6~8.1.[1] As an attempt to develop the superior sensing mechanism, we screen out promoter 170 (P170) and regulatory protein RcfB and introduce them into <span style="font-style: italic">E.coli</span> for adaption. </p></br>
+
  
<p>P170, a strongly acid-inducible promoter from <span style="font-style: italic">Lactococcus lactis</span>, is up-regulated at low pH during the transition to stationary phase.[2] The trans-acting protein RcfB, however, is involved in basal activity of P170 and is also essential for pH induction. The protein RcfB, upon activation by lactate, bind a DNA recognition motif within a promoter region and activates transcription.[3] When the cells are exposed to acid environments, the RcfB activation by an ‘acid’ signal allows its binding to the ACiD-box, resulting in transcription activation.[3] </p></br>
 
  
<div class="kuang" style="width:470px">
+
<div class="kuang" style="width:770px">
</br><a href="https://static.igem.org/mediawiki/2015/0/09/Lactate_activite.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/0/09/Lactate_activite.png"  width="450"  alt=""/></a>
+
</br><a href="https://static.igem.org/mediawiki/2015/3/31/Result_13%27.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/3/31/Result_13%27.png"  width="750"  alt=""/></a>
 
<div id="Enlarge">           
 
<div id="Enlarge">           
<p> <b>Figure 1.</b> <span style="font-size: 14px"> 图要改 111</span>
+
<p> <b>Figure 13.</b> <span style="font-size: 14px">(a) The comparison of polarization curve and power curve among <span style="font-style: italic">Shewanella</span>, <span style="font-style: italic">Shewanella</span> + MG1655, <span style="font-style: italic">Shewanella</span> + Δ<span style="font-style: italic">pflB</span> <span style="font-style: italic">ldhE</span> + Rf02S.
<a href="https://static.igem.org/mediawiki/2015/0/09/Lactate_activite.png"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
+
(b)The comparison of polarization curve and power curve among <span style="font-style: italic">Shewanella</span>, <span style="font-style: italic">Shewanella</span> + MG1655 and <span style="font-style: italic">Shewanella</span> + Δ<span style="font-style: italic">pflB</span> <span style="font-style: italic">ldhE</span> + <span style="font-style: italic">B. Subtilis</span>.
 +
   
 +
</span>
 +
<a href="https://static.igem.org/mediawiki/2015/3/31/Result_13%27.png"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
 +
<p>It is obvious that the power output in three-strain MFC systems with a more complete division of labor are far greater than single-strain and two-strian MFCs.</p></br>
 +
It significantly improves the commensalism relations between electricigens and fermentation bacteria.</p></br>
  
<p>To test the idea that P170 is sensitive to acidity under the regulation of RcfB, we construct RFP gene into circuit containing P170 and RcfB. The RFP is expressed on the basis of P170 activation and RcfB which functions as a novel regulatory protein however, is controlled by a constitutive promoter J23100. High level of P170 activity required both RcfB and acidic conditions. Therefore, with the constitutive expression of RcfB, once the pH reaches the threshold that usually ranges from pH 6.0 to pH 6.5, P170 will be induced so that RFP can reflect red fluorescence under UV detection. Although RFP verification test is well developed, other functional genes are also replace RFP gene as a strategy for application extension. In our later experiment, we substitute RFP for T7 polymerase for regulatory purpose. </p></br>
 
  
<div class="kuang" style="width:520px">
+
<div class="kuang" style="width:770px">
</br><a href="https://static.igem.org/mediawiki/2015/5/56/P170.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/5/56/P170.png"  width="500"  alt=""/></a>
+
</br><a href="https://static.igem.org/mediawiki/2015/d/d5/Result_14%27.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/d/d5/Result_14%27.png"  width="750"  alt=""/></a>
 
<div id="Enlarge">           
 
<div id="Enlarge">           
<p> <b>Figure 1.</b> <span style="font-size: 14px"> XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX</span><a href="https://static.igem.org/mediawiki/2015/5/56/P170.png"  target="_blank"><img src="https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg" width="20" height="20" align="right" alt="" /></a></p></div></div></br>
+
<p> <b>Figure 14.</b> <span style="font-size: 14px"> The comparison of polarization curve and power curve among <span style="font-style: italic">Shewanella</span> + Δ<span style="font-style: italic">pflB</span> <span style="font-style: italic">ldhE</span> + <span style="font-style: italic">B. Subtilis</span> and <span style="font-style: italic">Shewanella</span> + Δ<span style="font-style: italic">pflB</span> <span style="font-style: italic">ldhE</span> + Rf02S.</span>
 +
<a href="https://static.igem.org/mediawiki/2015/d/d5/Result_14%27.png"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>
  
<p style="font-weight: bolder">2  Regulating</p>
 
<p>After activated sensing system which determines the pH threshold to suppress bacterial population density, we need to figure out how to retrieve to the proper acidity for living. Here comes our regulating system for acid recovery. </p></br>
 
  
<p>The ssrA tag sequence (mf-ssrA tag) and the Lon protease (mf-Lon) from <span style="font-style: italic">Mesoplasma florum</span> are made up of the crucial elements of a protein degradation system. Experiments has been identified that mf-ssrA tag is efficiently recognized by mf-Lon where the tag appends to C terminus of native or denatured proteins resulted in their rapid proteolysis by mf-Lon.[4] Besides, degradation system based on the Gram-positive <span style="font-style: italic">M. florum</span> tmRNA system does not rely on host degradation systems and can function in a wide range of bacteria, which makes it an adaptive method in variety. Here, we use this proteolysis system for lactate regulation and keep it in a oscillatory value of acidity. </p></br>
 
  
<p>Specifically, we utilize the effect of mf-Lon as well as ldhE bearing with tag under the control T7 promoter and J23100 promoter respectively, to degrade the overmuch lactate. We also aware that T7 promoter activation requires the combination of T7 RNA polymerase which is extremely promoter-specific and transcribes only DNA downstream of a T7 promoter.[5] According to the extensive study of our ‘sensing’ system, we substitute the RFP for T7 polymerase hoping that lower pH stage can activate T7 polymerase activation which then combines with T7 promoter for acting and mf-Lon, in this way, also expresses. It turns out that <span style="font-style: italic">ldhE</span> togethered with mf-ssrA tag can be degraded by cytoplasmic mf-Lon. So the pH can maintain in a proper level for cells’ living. </p></br>
 
<div class="kuang" style="width:470px">
 
</br><a href="https://static.igem.org/mediawiki/2015/9/94/Mf-lon_biobrick.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/9/94/Mf-lon_biobrick.png"  width="450"  alt=""/></a>
 
<div id="Enlarge">         
 
<p> <b>Figure 1.</b> <span style="font-size: 14px"> 图要改  111</span>
 
<a href="https://static.igem.org/mediawiki/2015/9/94/Mf-lon_biobrick.png"  target="_blank"><img src=" https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg " width="20" height="20" align="right" alt="" /></a></p></div></div></br>(实验表征结果和说明)
 
<p>Notably, the proteolysis system has potential to apply in many fields. As a strategy to make it a toolbox for further research, we use different tags to test the strength of its proteolysis. With the reflection of red fluorescence, we are able to detect mf-Lon activity on various tags based on the distinguish value of UV detection. This toolbox is intended to be a fast and convenient method for prospective proteolysis option and degradation research. </p></br>
 
  
<div class="kuang" style="width:520px">
+
   
</br><a href="https://static.igem.org/mediawiki/2015/d/db/TAG.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/d/db/TAG.png"  width="500"  alt=""/></a>
+
   
<div id="Enlarge">         
+
   
<p> <b>Figure 1.</b> <span style="font-size: 14px"> XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX</span><a href="https://static.igem.org/mediawiki/2015/d/db/TAG.png"  target="_blank"><img src="https://static.igem.org/mediawiki/2013/9/90/Enlarge.jpg" width="20" height="20" align="right" alt="" /></a></p></div></div></br>
+
(实验表征结果和说明)
+
<h3>References</h3>
+
<hr></br>
+
<p> <span style="font-weight: lighter ;font-size:14px;" >
+
[1] Min C, Chung H, Choi W, et al. Linear correlation between inactivation of <span style="font-style: italic">E. coli</span> and OH radical concentration in TiO<sub>2</sub> photocatalytic disinfection.[J]. Water Research, 2004, 38(4):1069–1077. </br>
+
[2] Madsen S M, Arnau J, Vrang A, et al. Molecular characterization of the pH-inducible and growth phase-dependent promoter P170 of <span style="font-style: italic">Lactococcus lactis</span>.[J]. Molecular Microbiology, 1999, 32(1):75-87.</br>
+
[3] Madsen, Søren M, Hindré, Thomas, Le Pennec, Jean‐Paul, et al. Two acid‐inducible promoters from <span style="font-style: italic">Lactococcus lactis</span> require the cis‐acting ACiD‐box and the transcription regulator RcfB[J]. Molecular Microbiology, 2005, 56(3):735-746.</br>
+
[4] Eyal G, Sauer R T. Evolution of the ssrA degradation tag in <span style="font-style: italic">Mycoplasma</span>: specificity switch to a different protease.[J]. Proceedings of the National Academy of Sciences, 2008, 105(42):16113-16118.</br>
+
[5] Martin C T, Esposito E A, Theis K, et al. Structure and function in promoter escape by T7 RNA polymerase.[J]. Prog Nucleic Acid Res Mol Biol, 2005, 80(80):323–347.</p></br>
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  <div id="foot"><p>E-mail: ggjyliuyue@gmail.com |Address: Building No.20, No.92 Weijin road, Tianjin University, China | Zip-cod: 300072</br>
 
  <div id="foot"><p>E-mail: ggjyliuyue@gmail.com |Address: Building No.20, No.92 Weijin road, Tianjin University, China | Zip-cod: 300072</br>
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       <p>Copyright 2015@TJU iGEM Team</p></div>
 
       <p>Copyright 2015@TJU iGEM Team</p></div>
 
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Latest revision as of 14:03, 16 November 2015


Results


1 Lactate producing system



In order to verify our ldhE part and pflB knockout strategy have works well, we get following results.



Figure 1. a: The growth curve in anaerobic condition (M9 medium). b: Glucose consumption curve in anaerobic condition (M9 medium).


Shown as figure 1a, under anaerobic conditions, wild-type MG1655 has the same growth rate as MG1655ΔpflB, but MG1655ΔpflB + ldhE keeps relatively lower growth rate than the two strains mentioned before. Shown as figure 1b, the anaerobic glucose consumption rate keeps the nearly same among MG1655, MG1655ΔpflB and MG1655ΔpflB + ldhE.



Figure 2. a: The lactate production curve in anaerobic condition (M9 medium). b: The lactate production in anaerobic condition (M9 medium, 25h after inoculation)


In anaerobic environment, lactate production of MG1655, MG1655ΔpflB and MG1655ΔpflB+ldhE has large differences between each other. Notably, knocking out of pflB together with ldhE insertion will increase lactate generation up to ~3 g/L and the amount produced by MG1655ΔpflB is smaller than MG1655ΔpflB+ldhE.


So, knocking out of pflB can dramatically improve the lactate production. Besides, ldhE also plays an important role in lactate increase. Engineered lactate producing strain (MG1655ΔpflB+ldhE) has a greater utilization of carbon sources and a higher yield of lactate with M9 medium under the same concentrations of glucose.


2 Flavins producing system



To prove the EC10 and EC10* work as expected, we conduct several experiments and the results are as follows.



Figure 3. The yield of riboflavin in different strains: EC10, Rf02S (Δpgi + EC10), EC10*


As shown in figure 4, E. coli BL21 with EC10 reaches a final yield of 17 mg/L in flask cultivation. E. coli BL21Δpgi + EC10 reaches 33 mg/L. E. coli BL21 + EC10* reaches 90 mg/L.



Figure 4. The yield of riboflavin in different strains :EC10, Rf02S (Δpgi+EC10), EC10*


3 Co-culture MFC -- Division Of Labor




Figure 5. The electrical output of MFCs of single strain Shewanella oneidensis MR-1 with different substrates.


From the diagram above, we demonstrate that:

1. Shewanella cannot use glucose as a mere carbon substrate to generate power and so the potential keeps no more than 50 mV.

2. Shewanella can produce the maximal potential up to 150mV with supplement of sodium lactate.

3. Shewanella can produce even higher potential when we take sodium lactate as the carbon source and add little riboflavin into the system.


Conclusion: According to our experiment, our system is feasible in principle. Lactate can serve as the entry point in material flow and riboflavin as the major factor in energy and information flow, which lays a foundation in the later experiment design. We can use fermentation bacteria to provide carbon sources and electron shuttles, which subsequently, will increase the power generation capability in the co-culture system.



Figure 6. The electrical output of co-culture MFCs with preliminary division of labor


Description: The results show that dividing material, energy and information flow into separated fermentation bacteria is much better than combining the two tasks into one kind of bacteria. We suppose that the production of riboflavins and lactate cannot be consistent in single strain because lactate is the primary metabolites and riboflavin is the secondary.

However, the electricity output cannot sustain for long and decrease promptly (as shown in figure 5).Based on the measurement, samples from anode reaches an excessively low pH (lower than 6.2) which might not meet the survival requirement of Shewanella.

To optimize the system, we try to control the rate of lactate production and adjust the proportion of fermentation bacteria.



Figure 7. The electrical output of co-culture MFCs with optimized division of labor and bacteria proportion.


The maximal potential increased up to 322 mV and could maintain for 30 hours in the optimized three-strain system. (consist of Shewanella and two kinds of E. coli)



Figure 8. The electrical output of co-culture system with fermentation bacteria added 8h later into the system



Figure 12. a (1) A series of voltage brought by different external resistance from Shewanella + ΔpflB ldhE + Rf02S MFC system (2)The polarization curve and power density of Shewanella + ΔpflB ldhE + Rf02S MFC system. Besides, the highest power density can reach to 10mW/m2. b (1)A series of voltage brought by different external resistance from Shewanella + ΔpflB ldhE + B. subtilis (2)The polarization curve and power density of Shewanella + ΔpflB ldhE + B. subtilis MFC system.


The relations between current density and voltage is referred as polarization curve while the power curve can represent the relations between output power density and current density. We can learn from the figure 13, the highest power density of (1) reaches 10 mW/m2 and (2) reaches 17 mW/m2.



Figure 13. (a) The comparison of polarization curve and power curve among Shewanella, Shewanella + MG1655, Shewanella + ΔpflB ldhE + Rf02S. (b)The comparison of polarization curve and power curve among Shewanella, Shewanella + MG1655 and Shewanella + ΔpflB ldhE + B. Subtilis.


It is obvious that the power output in three-strain MFC systems with a more complete division of labor are far greater than single-strain and two-strian MFCs.


It significantly improves the commensalism relations between electricigens and fermentation bacteria.



Figure 14. The comparison of polarization curve and power curve among Shewanella + ΔpflB ldhE + B. Subtilis and Shewanella + ΔpflB ldhE + Rf02S.