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| | #body-PART{width:1100px;margin-left:auto;margin-right:auto;height:auto;background-color: #FFF;} | | #body-PART{width:1100px;margin-left:auto;margin-right:auto;height:auto;background-color: #FFF;} |
| | #body-PART-head{width:1100px;background-color:#FFF;font-family: "Lucida Grande", "Lucida Sans Unicode", "Lucida Sans", "DejaVu Sans", Verdana, sans-serif;color:rgba(204,204,255,1);font-size: 50px;float: left;text-align:center} | | #body-PART-head{width:1100px;background-color:#FFF;font-family: "Lucida Grande", "Lucida Sans Unicode", "Lucida Sans", "DejaVu Sans", Verdana, sans-serif;color:rgba(204,204,255,1);font-size: 50px;float: left;text-align:center} |
| − | #body-PART-l{width:22.2%;height:15500px;background-color:#FFF;float:left;} | + | #body-PART-l{width:22.2%;height:9100px;background-color:#FFF;float:left;} |
| | #body-PART-r{width:76.8%;height:auto;background-color:#FFF;float:left;margin-top:10px;margin-left:1%;text-align: justify;font-size:16px;font-family:Arial;line-height:150%;} | | #body-PART-r{width:76.8%;height:auto;background-color:#FFF;float:left;margin-top:10px;margin-left:1%;text-align: justify;font-size:16px;font-family:Arial;line-height:150%;} |
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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> |
| | + | </li> |
| | + | <li> |
| | + | <a href="https://2015.igem.org/Team:TJU/Results#Flavins producing system">Flavins producing system</a> |
| | + | </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> |
| − | <div align="center"><h2>Design</h2></div> | + | <div align="center"><h2>Results</h2></div> |
| | </br> | | </br> |
| − | <p>Naturally, we can barely find microorganisms that live in isolated niches, instead, there are full of various material, energy and information communications. In our project, we have been aware that <span style="font-style: italic">Shewanella</span> can efficiently use lactate as carbon substrate for power production. Therefore, high-yield of lactate seems critical for current generation and also for a reliable interaction between different species. Moreover, flavins mediated EET pathway is able to directly affect the electrons transfer and output, which inspired us to adopt flavins as a factor to achieve information interaction. Based on the research above, we further developed and optimized a co-culture system with three strains of bacterium in the MFC to achieve a more robust and efficient system. Apart from that, we designed a pH sensing and proteolysis system as a practical tool for troubleshooting and an attempt to benefit the future researchers. </p></br>
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| − | <div class="kuang" style="width:700px">
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| − | </br><a href="https://static.igem.org/mediawiki/2015/e/ee/Background_11.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/e/ee/Background_11.png" width="680" alt=""/></a>
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| − | <p> <b>Figure 1.</b> <span style="font-size: 14px"> The overall relationship of three kinds of bacteria in our co-culture system.</span><a href="https://static.igem.org/mediawiki/2015/e/ee/Background_11.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 name="Lactate producing system" id="Lactate producing system"></a><h3>1 Lactate producing system </h3> <hr></br> | | <a name="Lactate producing system" id="Lactate producing system"></a><h3>1 Lactate producing system </h3> <hr></br> |
| − | <p>Generally material flow, information flow, together with energy flow are key factors to regulate relations among bacterial consortium. Material flow, in particular, known as the most reliable and convenient method, is adopted in our construction. Typically the substances of the flow are often necessary nutrition for bacterial survival such as essential amino acid and carbon sources. </p></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> |
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| − | <div class="kuang" style="width:420px"> | + | <div class="kuang" style="width:770px"> |
| − | </br><a href="https://static.igem.org/mediawiki/2015/ e/ e6/% E7%A2%B3%E6%BA%90%EF%BC%88%E5%B8%8C%E7%93%A6%E6%B0%8F%EF%BC%89.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/ e/ e6/% E7%A2%B3%E6%BA%90%EF%BC%88%E5%B8%8C%E7%93%A6%E6%B0%8F%EF%BC%89.png" width=" 400" alt=""/></a> | + | </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> |
| | <div id="Enlarge"> | | <div id="Enlarge"> |
| − | <p> <b>Figure 2.</b> <span style="font-size: 14px"> The raletively narrow range of carbon source of <span style="font-style: italic">Shewanella</span>.</span><a href="https://static.igem.org/mediawiki/2015/ e/ e6/% E7%A2%B3%E6%BA%90%EF%BC%88%E5%B8%8C%E7%93%A6%E6%B0%8F%EF%BC%89.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 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> |
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| − | <p><span style="font-style: italic">Shewanella oneidensis</span> can generate electricity with a relatively narrow range of carbon substrates, including lactate, acetate, formate, pyruvate and some amino acid.[1] It has been shown that <span style="font-style: italic">Shewanella oneidensis</span> MR-1 prefers the utilization of lactate as an energy-favorable carbon substrate as lactate-based biomass yield was higher than that for either acetate or pyruvate. Similarly, the lactate-based growth rate was much higher as well.[2] Thus, lactate is the most suitable carbon source and also the key mediator for the material flow in our system. </p></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> |
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| − | <div class="kuang" style="width:670px">
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| − | </br><a href="https://static.igem.org/mediawiki/2015/c/c1/碳源（新体系1）.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/c/c1/碳源（新体系1）.png" width="650" alt=""/></a>
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| − | <p> <b>Figure 3.</b> <span style="font-size: 14px"> Our design of broadening the spectrum of carbon sources for MFCs based on <span style="font-style: italic">Shewanella</span>. Through it, we will presents the prospect of its expanding application range.</span><a href="https://static.igem.org/mediawiki/2015/c/c1/碳源（新体系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>
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| − | <p>In order to develop a proper mechanism for lactate supply, we use <span style="font-style: italic">Escherichia coli</span>, a well characterized bacterium, to produce lactate. Previously, researchers has developed a system that was able to produce 142.2 g/L of L-lactate with no more than 1.2 g/L of by-products accumulated by knocking out <span style="font-style: italic">ldhA</span> and <span style="font-style: italic">lldD</span> and inserting <span style="font-style: italic">L-LDH</span>.[3] Consequently, <span style="font-style: italic">E.coli</span> has many advantages as a host for production of lactic acid, including the ability to produce optically pure lactate, rapid growth under both aerobic and anaerobic conditions, and its simple nutritional requirements.[4] </p></br>
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| − | <p><span style="font-style: italic">Escherichia coli</span> grows fermentatively in glucose-containing medium under anaerobic condition with formation of a mixture of organic acids (lactate, acetate, formate and succinate) and ethanol, and lactate only takes no more than 50% of the total metabolites.[4] Since we need to provide enough material support for the growth and metabolism of <span style="font-style: italic">Shewanella</span> under anaerobic condition, we decide to enhance the yield of lactate by genetic engineering through two strategies: </p></br>
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| − | <p style="font-weight: bolder">1.1 ldh- Lactate dehydrogenase(LDHE) </p>
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| − | | + | |
| − | <p>LDH is an enzyme found in nearly all living cells (animals, plants, and prokaryotes), which catalyzes the conversion of pyruvate to lactate with NADH serving as the coenzyme. Naturally, <span style="font-style: italic">E.coli</span> can produce D-lactate by itself but the amount and the activity of lactate dehydrogenase becomes the bottleneck. We found that those two factors of enzyme are hard to change dramatically <span style="font-style: italic">in vivo</span>. So in our project, we intend to introduce high-yield exogenous L-(+)-lactate dehydrogenase gene (<span style="font-style: italic">ldhA</span>) from <span style="font-style: italic">Lactobacillus</span>, which can convert the redundant pyruvate and subsequently provide even more sustenance for <span style="font-style: italic">Shewanella</span>. Similar metabolic pathway has been found in a variety of organisms, to distinguish the differences, we renamed the heterogenous L-lactate dehydrogenase gene <span style="font-style: italic">ldhE</span> while homogenous one keeps original <span style="font-style: italic">ldhA</span>. </p></br>
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| − | <div class="kuang" style="width:470px">
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| − | </br><a href=" https://static.igem.org/mediawiki/2015/c/c8/Ldhe_gene.png" target="_blank" ><img src= " https://static.igem.org/mediawiki/2015/c/c8/Ldhe_gene.png" width="450" alt=""/></a>
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| − | <div id="Enlarge">
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| − | <p> <b>Figure 4.</b> <span style="font-size: 14px"> The part we design to produce L-lactate(LB medium).</span><a href=" https://static.igem.org/mediawiki/2015/c/c8/Ldhe_gene.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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| − | <p>After we transferred our part into <span style="font-style: italic">E.coli</span> BL21, we found there was little difference between the experimental group and control group.</p></br>
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| − | <div class="kuang" style="width:470px">
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| − | </br><a href="https://static.igem.org/mediawiki/parts/2/2b/LdhE-3.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/parts/2/2b/LdhE-3.png" width="450" alt=""/></a>
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| − | <div id="Enlarge">
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| − | <p> <b>Figure 5.</b> <span style="font-size: 14px"> The yield of lactate in flask-shaking fermentation condition (LB medium) </span>
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| − | <a href="https://static.igem.org/mediawiki/parts/2/2b/LdhE-3.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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| − | <p>In order to enhance the transformation efficiency from glucose to lactate, as well as improve the characterization of our <span style="font-style: italic">ldhE</span> part, we decide to knockout the the <span style="font-style: italic">pflB</span> and <span style="font-style: italic">poxB</span>.</p></br>
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| − | <div class="kuang" style="width:370px">
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| − | </br><a href="https://static.igem.org/mediawiki/2015/1/19/LdhE.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/1/19/LdhE.png" width="350" alt=""/></a>
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| − | <div id="Enlarge">
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| − | <p> <b>Figure 6.</b> <span style="font-size: 14px"> Lactate metabolic pathway. The <span style="font-style: italic">pflB</span> and <span style="font-style: italic">poxB</span> knocking out and <span style="font-style: italic">ldhE</span> insertion will result in the accumulation of lactate.
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| − | </span>
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| − | <a href="https://static.igem.org/mediawiki/2015/1/19/LdhE.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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| − | <p style="font-weight: bolder">1.2 <span style="font-style: italic">pflB</span> knockout</p>
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| − | <p>PFL is a crucial enzyme in the glucose metabolism under anaerobic condition, which can catalyze one molecule of pyruvate to one molecule of formicate and one molecule of acetylcoenzyme A (AcCoA). According to metabolic pathway, pyruvate is consumed both in the PFL and LDH reactions. In the wild type <span style="font-style: italic">E. coli</span>, LDH reaction is not as competitive as the reaction through PFL, and therefore, knockout of the PFL related genes will contribute to redistribute the metabolic flux. When the PFL pathway is blocked, <span style="font-style: italic">E. coli</span> will conceivably alter the distribution of these products to overcome the imbalanced reducing equivalents caused by the pathway knockout. Therefore, knockout of <span style="font-style: italic">pflB</span> not only decreased the carbon flow to acetyl-CoA under anaerobic condition but also reduced the anaerobic consumption of NADH through reductive TCA. [5] </p></br>
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| − | <p>After the knockout of <span style="font-style: italic">pflB</span>, though the growth rate was slightly slowed down, the yield of lactate of engineered strain was improved greatly and showed a significant difference from the wild type MG1655. From the result, we can see our part functioned as expected.</p></br>
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| | <div class="kuang" style="width:770px"> | | <div class="kuang" style="width:770px"> |
| − | </br><a href=" http://2https://static.igem.org/mediawiki/2015/ a/ a6/ 屏幕快照_2015-09-16_下午11.16.03.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/ a/ a6/ 屏幕快照_2015-09-16_下午11.16.03.png" width="750" alt=""/></a> | + | </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 7.</b> <span style="font-size: 14px"> Comparison diagram of lactate production of three different strains. MG1655: wild type ; MG1655Δ<span style="font-style: italic">pflB</span>: <span style="font-style: italic">pflB</span> knockout, <span style="font-style: italic">ldhE</span> blank; MG1655Δ<span style="font-style: italic">pflB</span>+<span style="font-style: italic">ldhE</span>: containing the functional <span style="font-style: italic">ldhE</span> part we design </span> | + | <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/ a/ a6/ 屏幕快照_2015-09-16_下午11.16.03.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 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> |
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| | + | <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> |
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| − | <p style="font-weight: bolder">1.3 Knockout strategy</p> | + | <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> |
| − | <p>For genes knockout, we adopted a effective, easy-to-use two-step system in which the cell is first transformed with a helper plasmid harboring genes encoding the λ-Red enzymes, I-SceI endonuclease, and RecA. λ-Red enzymes expressed from the helper plasmid are used to recombineer a small ‘landing pad’, a tetracycline resistance gene (<span style="font-style: italic">tetA</span>) flanked by I-SceI recognition sites and landing pad regions, into the desired location in the chromosome. After tetracycline selection for successful landing pad integrants, the cell is transformed with a donor plasmid carrying the desired insertion fragment; this fragment is excised by I-SceI and incorporated into the landing pad via recombination at the landing pad regions.[6] </p></br>
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| − | <p>First round of recombineering consists of three rounds of PCR for the construction of recombinant genome cassettes which subsequently positively selected by selection markers such as Tetr. In the second step, TetA marker was released by simultaneous induction of I-SceI and Red recombinase expression which serves as a negative selection.[7] (More details in Method.) </p></br>
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| − | <div class="kuang" style="width:770px">
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| − | </br><a href="https://static.igem.org/mediawiki/2015/4/48/Lambda_red.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/4/48/Lambda_red.png" width="750" alt=""/></a>
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| − | <p> <b>Figure 8.</b> <span style="font-size: 14px"> First step in the new two-step scar-less gene deletion using λ-Red recombineering method. Three rounds of PCR together with <span style="font-style: italic">tet</span> selection make up of our recombinant.</span>
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| − | <a href="https://static.igem.org/mediawiki/2015/4/48/Lambda_red.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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| − | </br>
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| | <a name="Flavins producing system" id="Flavins producing system"></a><h3> 2 Flavins producing system </h3> <hr></br> | | <a name="Flavins producing system" id="Flavins producing system"></a><h3> 2 Flavins producing system </h3> <hr></br> |
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| − | <p><span style="font-style: italic">Shewanella oneidensis</span> MR-1, a facultative anaerobe, has been widely used as a model anode biocatalyst in microbial fuel cells (MFCs) due to its easiness of cultivation, adaptability to aerobic and anaerobic environment and both respiratory and electron transfer versatility. [8] </p></br> | + | <p>To prove the EC10 and EC10* work as expected, we conduct several experiments and the results are as follows.</p></br> |
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| − | | + | <div class="kuang" style="width:570px"> |
| − | <p>Particularly, EET pathway can be subdivided into direct EET and mediated EET and the latter one limits the efficiency of electron transfer between bacteria and electrode due to the deficiency of electron mediator. To regulate relations of energy and information in the consortium, flavins hold the key to success. </p></br>
| + | </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> |
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| − | <p>It is widely accepted that interfacial EET is the rate-limiting step in the EET processes which can be relieved by some redox active molecules such as quinines and metal-centered porphyrin-ring derivatives. However, those redox active molecules are generally costly and toxic to anodic electricigens. Besides, <span style="font-style: italic">Shewanella</span> is capable of utilizing self-secreted flavins like riboflavin (RF) to accelerate EET, which is much more efficiently than other exogenous active molecules. More specifically, riboflavin (RF) and flavin mononucleotide (FMN) enhance EET more than five times, with much lower concentrations than those needed for anthraquinone-2,6-disulfonate (AQDS) shuttling. So we choose flavins as one entry point to enhance the electricity output of MFCs. [9] </p></br>
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| − | <div class="kuang" style="width:620px"> | + | |
| − | </br><a href=" https://static.igem.org/mediawiki/2015/ 6/ 61/ EET. png" target="_blank" ><img src= " https://static.igem.org/mediawiki/2015/ 6/ 61/ EET. png" width=" 600" alt=""/></a> | + | |
| | <div id="Enlarge"> | | <div id="Enlarge"> |
| − | <p> <b>Figure 9.</b> <span style="font-size: 14px"> Comparison of co-factor EET model(a) and diffusion EET model(b), (b) also reveals the electron pathway by endogenous flavins </span> | + | <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/2015/6/61/EET.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 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> |
| − | | + | |
| − | | + | |
| − | <p>In EET model, outward current flows from interior of cells to outer membrane (OM) and extracellular anodes through a metal-reducing conduit (Mtr pathway), where electrons (from NADH, the intracellular electron carrier) flow through the menaquinol pool, CymA (inner membrane [IM] tetraheme c-type cytochromes [c-Cyts]), MtrA (periplasmic decaheme c-Cyts), MtrB (β-barrel trans-OM protein) and finally to MtrC and OmcA (two OM decaheme c-Cyts). However, the principle of how flavins function still remains controversial. [10] </p></br>
| + | |
| − | | + | |
| − | <p>Traditional model demonstrates that flavins carry the electrons from OM c-Cyts to electrode by diffusion, which has been in debate due to thermodynamic disproof. Recently, another interfacial EET model was proposed, where flavin may serve as a co-factor binding to OmcA or MtrC to dictate EET. Fully oxidized flavin (Ox) accepts one electron from reduced heme of OM c-Cyts, and binds to OM c-Cyts as a cofactor in the semiquinone (Sq) form with shifted potential. Ox/Sq redox cycling in OM c-Cyts donates electrons to electrodes in a one electron reaction mode via direct contact.[10] </p></br>
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| − | <div class="kuang" style="width:420px">
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| − | </br><a href="https://static.igem.org/mediawiki/2015/2/29/Riboflavin_metabolism.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/2/29/Riboflavin_metabolism.png" width="400" alt=""/></a>
| + | |
| − | <div id="Enlarge">
| + | |
| − | <p> <b>Figure 10.</b> <span style="font-size: 14px"> Pathway for engineered riboflavin and FMN production <span style="font-style: italic">E. coli</span>. </span>
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| − | <a href=" https://static.igem.org/mediawiki/2015/2/29/Riboflavin_metabolism.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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| − | <p>Although <span style="font-style: italic">Shewanella</span> uses endogenous flavins to mediate electrons, the amount of its production is deficient and multi-tasks brought by self-engineering may also reduce the transfer efficiency. As a consequence, we had two strategies to construct the zymophyte and further improve the production. Firstly, we introduced flavin producing genes using the <span style="font-style: italic">E.coli</span> as chassis. Secondly, we get an engineered <span style="font-style: italic">B.subtilis</span> strain from the lab of Dr. Tao Chen [11](more detail in attribution) </p></br>
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| − | | + | |
| − | <p>Based on the EET pathway theory, we suggest that by maximizing the amount of flavins, we may significantly enhance the EET efficiency and achieve a relatively high power generation.</p></br>
| + | |
| − | | + | |
| − | <p>We found a part BBa_K1172303 in Part Registry constructed by 2013 Team Bielefelf-Germany which was also aimed at producing riboflavins. However, the gene cluster showed a maximum output of 6 mg/L even with a strong promoter, which was insufficient to maintain a high and constant efficiency of EET pathway in our co-culture system. So we decided to optimize the part BBa_K1172303 to enhance the production of riboflavins. </p></br>
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| − | <div class="kuang" style="width:770px">
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| − | </br><a href="https://static.igem.org/mediawiki/2015/7/73/EC10_GENE改.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/7/73/EC10_GENE改.png" width="750" alt=""/></a>
| + | |
| − | <div id="Enlarge">
| + | |
| − | <p> <b>Figure 11.</b> <span style="font-size: 14px"> Two different parts we designed to produce flavins. </span>
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| − | <a href="https://static.igem.org/mediawiki/2015/7/73/EC10_GENE改.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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| − | <p>As we learn from metabolic flux (figure 2), it reveals the relevant pathways of riboflavin production and engineering strategies for riboflavin production. In their previous research(Tao, et al) [7] ,they construct a high-yield <span style="font-style: italic">E.coli</span> strain with a yield of 229.1 mg/L. Based on their study, we constructed a flavin producing part (<span style="font-style: italic">ribABDEC</span> cluster) named EC10.(as shown in figure 11a). The part(<a href="http://parts.igem.org/Part:BBa_K1696011">BBa_K1696011</a>) we designed, compared with BBa_K117230, has been well optimized and the yield of that reached 17 mg/L.( as shown in figure 13). We can see from the results, the functionality of their parts has successfully improved. </p></br>
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| − | <div class="kuang" style="width:470px">
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| − | </br><a href="https://static.igem.org/mediawiki/parts/c/c2/Ribo-3.jpg" target="_blank" ><img src= "https://static.igem.org/mediawiki/parts/c/c2/Ribo-3.jpg" width="450" alt=""/></a>
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| − | <div id="Enlarge">
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| − | <p> <b>Figure 12.</b> <span style="font-size: 14px"> The production of EC10 in tubes.</span>
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| − | <a href="https://static.igem.org/mediawiki/ parts/ c/ c2/ Ribo-3.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> | + | |
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| − | <p>In the meanwhile, based on co-factor model, EET pathway points out that FMN plays a critical role in electron transfer. Through the study by Dr. Tao Chen, they have weakened the RBS upstream of <span style="font-style: italic">ribC</span> to divert more of the material flux to RF production.[7] Based on their viewpoint, we got further to design a strong RBS sequence upstream of the <span style="font-style: italic">ribC</span> and we rename the new part as EC10*(<a href="http://parts.igem.org/Part:BBa_K1696010">BBa_K1696010</a>). The yield of EC10* reached 90 mg/L(as shown in fugure 11b). The strong RBS before <span style="font-style: italic">ribC</span> sequence can lead to the FMN accumulation while riboflavin consumes a bit, which may in turn, result in increase both for riboflavin and FMN as a whole. </p></br> | + | <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> |
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| | <div class="kuang" style="width:590px"> | | <div class="kuang" style="width:590px"> |
| | </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> | | </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 13.</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> | + | <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/2015/2/25/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> | | <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> |
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| − | <p>When we characterized our part, we were not able to detect the FMN for lacking proper equipments. However, we were surprised to find that the production of EC10* was even better with a larger output of riboflavins. For riboflavin measurements, culture samples were diluted with 0.05 M NaOH to the linear range of the spectrophotometer and the A444 was immediately measured, according to the Dr. Tao Chen’s method [7]. As for the reason of production improvement of RF, we speculate that the strengthening of <span style="font-style: italic">ribC</span> gene can improve both the RF and FMN yield through the flavin metabolic pathway. </p></br>
| + | <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> |
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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;">Labor Division</span> </h3> <hr></br> | + | |
| − | <p>Microorganisms rarely live in isolated niches. One reason is that the maintenance of viability of these microbes may need supplementary metabolites or other signaling chemicals provided by other microbes in the ecosystems and communities.[11] Conforming to the trend of revolution, we apply the bacterial consortium in our MFC for greater power generation and increased duration. </p></br>
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| − | | + | |
| − | <p>Apart from the universal trend of nature, the mixed culture shares some unique advantages particularly in our MFC. For example, <span style="font-style: italic">Shewanella</span> originally can merely use few simple carbon substrates like lactate, acetate, formate, pyruvate and some amino acid.[1] However, our consortium as a whole can expand the absorption range of carbon resources and develop a more complex diagram of carbon input for <span style="font-style: italic">Shewanella</span>. Also, our intention is able to achieve comprehensive functions through <span style="font-style: normal;font-weight: bold; color:#ddbf73;">“Labor Division”</span> that monoculture can hardly manage. For instance, if we use only <span style="font-style: italic">Shewanella</span> to supply electricity, the production of flavins as well as other related reaction will inhibit current creation. These are fundamental reasons that we adopt the “mix” strategy in our project.</p></br>
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| − | <p style="font-weight: bolder">3.1 First Trial</p>
| + | |
| − | <p>Our experiment started with monoculture of <span style="font-style: italic">Shewanella</span>, in which we have found several important conclusions that might be used in the later trials: </br>
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| − | (i) <span style="font-style: italic">Shewanella</span> alone cannot take good use of glucose as the mere carbon source to generate power. </br>
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| − | (ii) Voltage has been improved when we add flavins to the monoculture medium, which proved that flavins indeed would help the power enhance. </br>
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| − | (iii) As an attempt to choose a proper substance being used in the experiment, we put sodium lactate into service and found that <span style="font-style: italic">Shewanella</span> could achieve power output. </p></p></br>
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| | <div class="kuang" style="width:770px"> | | <div class="kuang" style="width:770px"> |
| − | </br><a href="https://static.igem.org/mediawiki/2015/ 1/ 1b/ TJU-design-2.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/ 1/ 1b/ TJU-design-2.png" width="750" alt=""/></a> | + | </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 13.</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> | + | <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/ 1/ 1b/ TJU-design-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> | + | <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> |
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| − | <p style="font-weight: bolder">3.2 Second Trial: </p> | + | <p>From the diagram above, we demonstrate that：</p> |
| − | <p>Since we’ve constructed the lactate producing strain (ldhE), the flavin producing strain (EC10) and an engineered strain bearing both ldhE and EC10, we initiated our co-culture experiment with different labor division. </p></br> | + | <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"> | | <div class="kuang" style="width:770px"> |
| − | </br><a href="https://static.igem.org/mediawiki/2015/ 3/ 3a/ TJU-design-3.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/ 3/ 3a/ TJU-design-3.png" width="750" alt=""/></a> | + | </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 14.</b> <span style="font-size: 14px"> The electrical output of co-culture MFCs with preliminary labor division</span> | + | <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/ 3/ 3a/ TJU-design-3.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 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> |
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| − | <p>From the figure above, we found that the co-culture system with three different strains has a higher electricity output than two-strain system before 20h, showing that a more complete labor division may have a better effect in the co-culture system. It is because that we can more accurately control the amount and ratio of lactate and flavins when they are produced in separated strains. </p></br> | + | <p>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.</p> |
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| − | <div class="kuang" style="width:470px"> | + | <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> |
| − | </br><a href="https://static.igem.org/mediawiki/2015/3/39/Shizhi.jpg" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/3/39/Shizhi.jpg" width="450" alt=""/></a>
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| − | <div id="Enlarge">
| + | |
| − | <p> <b>Figure 15.</b> <span style="font-size: 14px">
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| − | pH test of anode samples, with only sample 1 get reached above 6.5 </span>
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| − | <a href="https://static.igem.org/mediawiki/2015/3/39/Shizhi.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>
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| − | <p>However, the electricity output dropped promptly after 30h. (as shown in figure 1). In the meanwhile, we took samples from anode finding that pH in anode was excessively low (lower than 6.2) which might not meet the survival requirement of <span style="font-style: italic">Shewanella</span>. </p></br> | + | <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 reason of the acidity, based on our analysis, could be the overmuch glucose or the high ratio of fermentation bacteria in the system. Particularly, our medium consist of 10 g/L glucose as well as 5% LB and 95% M9 medium. Both of the possibilities above can lead to the overproduction of lactate and other acid metabolites like formic acid which are hard to consume. </p></br>
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| − | <p>So, in order to solve the pH problem, we have two strategies. Firstly, we can optimize the medium component by reducing ratio of C and N. Secondly, we would optimize the proportion of the fermentation bacteria. </p></br>
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| − | | + | |
| − | <p>Apart from that, we also found that excessive portion of zymophyte would inhibit biofilm formation of <span style="font-style: italic">Shewanella</span> by zymophyte taking up the spaces in electrode. </p></br>
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| − | | + | |
| − | <p style="font-weight: bolder">3.3 Third trial</p>
| + | |
| − | <p>When we have preliminarily optimized the bacterial proportion and medium compositions, we got a significant improvement of the electricity output in spite of the relatively short sustaining time.(shown as follows) </p></br>
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| | <div class="kuang" style="width:770px"> | | <div class="kuang" style="width:770px"> |
| − | </br><a href="https://static.igem.org/mediawiki/2015/0/ 04/ TJU-design-4.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/0/ 04/ TJU-design-4.png" width="750" alt=""/></a> | + | </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"> | | <div id="Enlarge"> |
| − | <p> <b>Figure 16.</b> <span style="font-size: 14px"> The electrical output of co-culture MFCs with optimized labor division and bacteria proportion</span> | + | <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/ 04/ TJU-design-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> | + | <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> |
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| − | | + | <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> |
| − | <p>We also attempted to put the fermentation bacteria into the system some time after <span style="font-style: italic">Shewanella</span>. However, the later joining of fermentation bacteria will the stability of the system so that the output oscillated for a period of time. We can see from the figure**, even though the lasting time was longer than before, the maximum electricity output was still limited. </p></br> | + | |
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| | <div class="kuang" style="width:770px"> | | <div class="kuang" style="width:770px"> |
| − | </br><a href="https://static.igem.org/mediawiki/2015/ 1/ 15/ TJU-design-5.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/ 1/ 15/ TJU-design-5.png" width="750" alt=""/></a> | + | </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> |
| | <div id="Enlarge"> | | <div id="Enlarge"> |
| − | <p> <b>Figure 17.</b> <span style="font-size: 14px"> The electrical output of co-culture system with fermentation bacteria added 8 h later into the system</span> | + | <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/ 1/ 15/ TJU-design-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> | + | <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> |
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| − | <p>Subsequently, we arranged variable mixture for three-strain system to determine the best proportion for electricity generation. Besides, we continued to optimize the culture condition (95%M9, 5%LB and glucose 2 g/L), we can see from the figure that the three-strain system had a maximum output at around 350mV and had a significant advantage over the two-strain system. Additionally, the lasting time was over 80h without any supplementary glucose, which indicated the high utilization rate of carbon source and the relatively stable relationship among the three strains. </p></br>
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| | <div class="kuang" style="width:770px"> | | <div class="kuang" style="width:770px"> |
| − | </br><a href="https://static.igem.org/mediawiki/2015/ f/ f1/ TJU-design-6.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/ f/ f1/ TJU-design-6.png" width="750" alt=""/></a> | + | </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"> | | <div id="Enlarge"> |
| − | <p> <b>Figure 18.</b> <span style="font-size: 14px"> 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>+Rf02S, the three-strain co-culture system reached a relatively higher potential that kept around 350mV in 80 hours</span> | + | <p> <b>Figure 12.</b> <span style="font-size: 14px"> a |
| − | <a href="https://static.igem.org/mediawiki/2015/ f/ f1/ TJU-design-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> | + | (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 |
| | + | (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>. |
| | + | b |
| | + | (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. |
| | + | </span> |
| | + | <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> |
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| | + | <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> |
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| − | <p style="font-weight: bolder">3.4 Fourth trial</p>
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| − | <p>We innovatively introduced a third species, <span style="font-style: italic">Bacillus subtilis</span>, into our system. Considering <span style="font-style: italic">Bacillus subtilis</span> take KNO<sub>3</sub> as the substrates instead of glucose in anaerobic condition, we could reduce the competition for carbon sources between two kinds of fermentation bacteria. Also it has been reported that engineered <span style="font-style: italic">B.subtilis</span> could get a high output of flavins which is exactly what we want. So, we choosed <span style="font-style: italic">Bacillus subtilis</span> as another kind of fermentation bacteria. </p></br>
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| − | <p>It can been seen from the figure**, the electricity output of three-strain co-culture system with <span style="font-style: italic">B.subtilis</span> could reach more than 500mV enduring around 80h, showing a great difference from the <span style="font-style: italic">Shewanella</span> single-strain system. Compared with two-strain system below, the three-strain system with <span style="font-style: italic">B.subtilis</span> also showed a visible advantage. </p></br>
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| | <div class="kuang" style="width:770px"> | | <div class="kuang" style="width:770px"> |
| − | </br><a href="https://static.igem.org/mediawiki/2015/ 8/ 85/ TJU-design-7.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/ 8/ 85/ TJU-design-7.png" width="750" 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 19.</b> <span style="font-size: 14px"> The electrical output of single strain <span style="font-style: italic">Shewanella</span> compared to <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> co-culture system with glucose as carbon source.</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/ 8/ 85/ TJU-design-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> | + | (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> |
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| | <div class="kuang" style="width:770px"> | | <div class="kuang" style="width:770px"> |
| − | </br><a href="https://static.igem.org/mediawiki/2015/ 1/ 1c/ TJU-design-8.png" target="_blank" ><img src= "https://static.igem.org/mediawiki/2015/ 1/ 1c/ TJU-design-8.png" width="750" 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 20.</b> <span style="font-size: 14px"> The electrical 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> compared to <span style="font-style: italic">Shewanella</span>+Δ<span style="font-style: italic">plfB</span> <span style="font-style: italic">ldhE</span>+Rf02S with glucose and KNO<sub>3</sub> as substrates</span> | + | <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/ 1/ 1c/ TJU-design-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> | + | <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> |
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| − | <p style="font-weight: bolder">Future Work: </p>
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| − | <p>Although we made several innovative research in our project, there are still many other aspects to develop and optimize in the future. </br>
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| − | 1. We plan to find out biofilm parts of <span style="font-style: italic">E.coli</span> and modify several key point to reduce adhesion effect. </br>
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| − | 2. We need to further adjust ratio of our three-strain system to bring a quicker speed toward maximum. </br>
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| − | 3. Keeping power generation unchanged, we can reduce the volume of our device so that it will increase current density. </br>
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| − | 4. Making improvements in <span style="font-style: italic">Shewanella</span> for more electricity output. We intend to weaken any other unrelated functions except for power production in <span style="font-style: italic">Shewanella</span> and using co-culture system to produce necessary nutrition for its survival. </p></br>
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| − | <a name="References" id="References"></a><h3>References</h3>
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| − | <hr></br>
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| − | <p> <span style="font-weight: lighter ;font-size:14px;" > [1] Tang Y J, Meadows A L, Keasling J D. A kinetic model describing <span style="font-style: italic">Shewanella oneidensis</span> MR-1 growth, substrate consumption, and product secretion.[J]. Biotechnology & Bioengineering, 2007, 96(1):125–133.</br>
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| − | [2] Feng X, Xu Y, Chen Y, et al. Integrating Flux Balance Analysis into Kinetic Models to Decipher the Dynamic Metabolism of <span style="font-style: italic">Shewanella oneidensis</span> MR-1[J]. Plos Computational Biology, 2011, 8(2):e1002376-e1002376. </br>
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| − | [3] Highly efficient L-lactate production using engineered <span style="font-style: italic">Escherichia coli</span> with dissimilar temperature optima for L-lactate formation and cell growth. </br>
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| − | [4] Liu H, Kang J, Qi Q, et al. Production of lactate in <span style="font-style: italic">Escherichia coli</span> by redox regulation genetically and physiologically.[J]. Appl Biochem Biotechnol, 2011, 164(2):162-169. </br>
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| − | [5] Zhu J, Shimizu K, Zhu J, et al. Effect of a single-gene knockout on the metabolic regulation in <span style="font-style: italic">Escherichia coli</span> for D-lactate production under microaerobic condition[J]. Metabolic Engineering, 2005, 7(2):104–115. </br>
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| − | [6] Thomas E, Kuhlman, Edward C, Cox. Site-specific chromosomal integration of large synthetic constructs.[J]. Nucleic Acids Research, 2010, 38(38). </br>
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| − | [7] Lin Z, Xu Z, Li Y, et al. Metabolic engineering of <span style="font-style: italic">Escherichia coli</span> for the production of riboflavin[J]. Microbial Cell Factories, 2014, 13(1):1-12.
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| − | </br>
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| − | [8] Wang X, Tao L, Wang H, et al. Improving mediated electron transport in anodic bioelectrocatalysis[J]. Chemical Communications, 2015. </br>
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| − | [9] Okamoto A, Nakamura R, Nealson K H, et al. Bound Flavin Model Suggests Similar Electron‐Transfer Mechanisms in <span style="font-style: italic">Shewanella</span> and <span style="font-style: italic">Geobacter</span>[J]. Chemelectrochem, 2014, 1(11):1808-1812. </br>
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| − | [10] Yang Y, Ding Y, Hu Y, et al. Enhancing Bidirectional Electron Transfer of <span style="font-style: italic">Shewanella oneidensis</span> by a Synthetic Flavin Pathway[J]. Acs Synthetic Biology, 2015. </br>
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| − | [11]Shi S, Chen T, Zhang Z, et al. Transcriptome analysis guided metabolic engineering of Bacillus subtilis for riboflavin production[J]. Metabolic engineering, 2009, 11(4): 243-252.</br>
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| − | [12] Song H, Ding M Z, Jia X Q, et al. Synthetic microbial consortia: from systematic analysis to construction and applications[J]. Chem.soc.rev, 2014, 43(20):6954-6981.</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> |
| | </p> | | </p> |
| | <p>Copyright 2015@TJU iGEM Team</p></div> | | <p>Copyright 2015@TJU iGEM Team</p></div> |
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