Difference between revisions of "Team:CHINA CD UESTC/Design"
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− | We mainly designed three vectors respectively carrying <i>mamW</i>+<i>RFP</i>+laccase , <i>mamAB</i> and <i>mamGFDC</i> + <i> | + | We mainly designed three vectors respectively carrying <i>mamW</i> |
+ | + <i>RFP</i> | ||
+ | + <i>laccase</i> , | ||
+ | <i>mamAB</i> | ||
+ | and | ||
+ | <i>mamGFDC</i> | ||
+ | + | ||
+ | <i>mms6</i> | ||
+ | + | ||
+ | <i>mamXY</i> | ||
+ | . Our purpose is to accomplish our magnetotactic | ||
+ | <i>E.coli</i> | ||
+ | and fix the laccase on the magnetosome membrane. Finally we put the magnetosomes carrying laccases into our enzymatic bio-fuel cell (EBFC). | ||
</p> | </p> | ||
</div> | </div> | ||
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<div class="grid_8"> | <div class="grid_8"> | ||
<p> | <p> | ||
− | This summer, CHINA_CD_UESTC team | + | This summer, CHINA_CD_UESTC team worked very hard in order to make a high-efficiency enzymatic biofuel cell (EBFC) by enriching the laccase on the cathode electrode. We transferred four operons-- |
− | + | <i>mamAB</i> | |
− | + | , | |
− | + | <i>mamGFDC</i> | |
− | + | , | |
− | + | <i>mamXY</i> | |
− | + | and | |
+ | <i>mms6</i> | ||
+ | , which were related to magnetosome formation into | ||
+ | <i>E.coli.</i> | ||
+ | the modified | ||
+ | <i>E.coli</i> | ||
+ | can produce laccase-carried magnetosome. Therefore, we could immobilize and enrich laccase on the cathode electrode by magnet. In our project, we improved previous <i>laccase</i> part ( | ||
+ | <a href="http://parts.igem.org/Part:BBa_K863005">BBa_K863005</a> | ||
+ | ) and made it visible. | ||
</p> | </p> | ||
+ | <P> | ||
+ | The EBFC schematic diagram as following is the final <strong>prototype</strong> | ||
+ | of our project(Figure 1): | ||
+ | </P> | ||
<div class="project_pic"> | <div class="project_pic"> | ||
<p id="pic_title"></p> | <p id="pic_title"></p> | ||
− | <img src="https://static.igem.org/mediawiki/2015/ | + | <img src="https://static.igem.org/mediawiki/2015/0/0b/CHINA_CD_UESTC_DESIGN_overview.png" width="70%"> |
− | <p id="pic_illustration"> | + | <p id="pic_illustration"> <strong>Figure 1</strong> |
− | + | . Schematic diagram of our EBFC. On the anode, glucose is oxidized to gluconolactone, where the electrons are transferred from the GOX to CNT. On the cathode, laccase is immobilized and enriched on the electrode by magnetosome. Electrons are transferred from CNT to laccase where dioxygen is reduced to water. | |
</p> | </p> | ||
</div> | </div> | ||
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<div id="content" class="grid_12"> | <div id="content" class="grid_12"> | ||
− | <h3> | + | <h3>Visible laccase in EBFC</h3> |
</div> | </div> | ||
<div class="clear"></div> | <div class="clear"></div> | ||
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<p> | <p> | ||
After a review of the relevant literature <sup>[1]</sup> | After a review of the relevant literature <sup>[1]</sup> | ||
− | , we learned that | + | , we learned that the laccase has advantages over other oxidases. Thereby, we chose the laccase as the enzyme for cathode. In order to make laccase visible, we designed a recombinant vector to fuse |
+ | <i>RFP</i> | ||
+ | with the <i>laccase</i>. And <i>laccase</i> gene was obtained from | ||
+ | <a href="http://parts.igem.org/Part:BBa_K863005">BBa_K863005</a> | ||
+ | on the 2015 Kit Plate2. While the | ||
+ | <i>RFP</i> | ||
+ | gene was taken from | ||
+ | <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> | ||
+ | on the 2015 Kit Plate3. We designed the vector piGEM-RL as below(Figure 2).After constructing the vectors completely, we detected whether it work or not by the method of ABTS <sup>[2]</sup> : | ||
</p> | </p> | ||
<div class="project_pic"> | <div class="project_pic"> | ||
<p id="pic_title"></p> | <p id="pic_title"></p> | ||
<img src="https://static.igem.org/mediawiki/2015/1/12/CHINA_CD_UESTC_DESIGN_LACCASE02.png" width="60%"> | <img src="https://static.igem.org/mediawiki/2015/1/12/CHINA_CD_UESTC_DESIGN_LACCASE02.png" width="60%"> | ||
− | <p id="pic_illustration"></p> | + | <p id="pic_illustration"> |
+ | <strong>Figure 2.</strong> | ||
+ | Schematic of piGEM-RL construction. Laccase: efficient oxidase, catalyzes the substrate to produce electrons and environmentally friendly. RFP: the reporter protein. | ||
+ | </p> | ||
</div> | </div> | ||
− | + | <p>The laccase can be used to construct a common EBFC as biological cathode. we conceived a prototype( Figure 3). In the EBFC, we used carbon paper which was full of carbon microfibers as electrode because it has good conductivity and large surface area. Glucose oxidase and RFP+laccase were chosed to catalyze reaction for producing electricity! | |
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<div class="project_pic"> | <div class="project_pic"> | ||
− | <p id="pic_title"> | + | <p id="pic_title"></p> |
− | + | <img src="https://static.igem.org/mediawiki/2015/f/f7/CHINA_CD_UESTC_DesignOverview.png" width="60%"> | |
− | + | <p id="pic_illustration"> | |
− | + | <strong>Figure 3.</strong> | |
− | < | + | Schematic diagram of our EBFC. On the anode, glucose is oxidized to gluconolactone, where the electrons are transferred from the GOX to CNT. On the cathode, electrons are transferred from CNT to laccase where dioxygen is reduced to water. |
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</p> | </p> | ||
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<p> | <p> | ||
− | + | The components of the EBFC are listed in the Table 1. | |
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</p> | </p> | ||
<div class="project_pic"> | <div class="project_pic"> | ||
<p id="pic_title"></p> | <p id="pic_title"></p> | ||
− | <img src="https://static.igem.org/mediawiki/2015/a/a8/CHINA_CD_UESTC-DesignPlus01.png" width=" | + | <img src="https://static.igem.org/mediawiki/2015/a/a8/CHINA_CD_UESTC-DesignPlus01.png" width="70%"> |
<p id="pic_illustration"></p> | <p id="pic_illustration"></p> | ||
</div> | </div> | ||
<p> | <p> | ||
− | + | The <strong>main materials</strong> of our EBFC. | |
+ | </p> | ||
+ | <p>The main materials of our EBFC(Figure 4). Carbon paper consists of carbon microfibers manufactured into flat sheets. It is used to help facilitates the reaction. Glucose oxidase oxidizes glucose into glucolactone. Electrons are released in the reaction.The fusion protein (RFP+laccase) transfers electrons to the terminal electron acceptor oxygen. | ||
</p> | </p> | ||
<div class="project_pic"> | <div class="project_pic"> | ||
<p id="pic_title"></p> | <p id="pic_title"></p> | ||
− | <img src="https://static.igem.org/mediawiki/2015/3/3b/CHINA_CD_UESTC-DesignPlus02.png" width=" | + | <img src="https://static.igem.org/mediawiki/2015/3/3b/CHINA_CD_UESTC-DesignPlus02.png" width="60%"> |
<p id="pic_illustration"> | <p id="pic_illustration"> | ||
− | + | <strong>Figure 4. (A)</strong> | |
+ | Carbon papers as the electrode. | ||
+ | <strong>(B)</strong> | ||
+ | Glucose oxidase on the anode. | ||
+ | <strong>(C)</strong> | ||
+ | RFP+laccase on the cathode. | ||
</p> | </p> | ||
</div> | </div> | ||
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<div id="content" class="grid_12"> | <div id="content" class="grid_12"> | ||
− | <h3> | + | <h3>Express magnetosome in <i>E.coli</i> for immobilization</h3> |
</div> | </div> | ||
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<div id="content"> | <div id="content"> | ||
<div class="grid_8"> | <div class="grid_8"> | ||
<p> | <p> | ||
− | In the magnetotactic bacteria, there are | + | In order to immobilize and enrich laccase on the cathode pole, we want to connect laccase with magnetosome. Then the magnetosome carrying laccase can be attracted by magnet. First, we need to obtain lots of magnetosomes. In the magnetotactic bacteria, there are four steps to generate magnetosome |
− | + | <sup>[3]</sup> | |
− | <sup>[ | + | :1-invagination, 2-protein localization, 3-initiation of crystal mineralization, 4-crystal maturation. There exist four operons-- |
− | :1-invagination, 2-protein localization, 3-initiation of crystal mineralization, 4-crystal maturation. | + | <i>mamAB</i> |
+ | , | ||
+ | <i>mamGFDC</i> | ||
+ | , | ||
+ | <i>mamXY</i> | ||
+ | and | ||
+ | <i>mms6</i> | ||
+ | , which are related to magnetosome formation. | ||
</p> | </p> | ||
− | < | + | <h4>1. <i>mamAB</i></h4> |
<p> | <p> | ||
− | + | This section describes the function of the vector piGEM-mamAB. It carries | |
+ | <i>mamAB</i> | ||
+ | operon whose length is up to 17kb. Previous studies have shown that | ||
+ | <i>mamAB</i> | ||
+ | operon is one of the four core operons related to magnetosomes formation | ||
+ | <sup>[4]</sup> | ||
+ | . For consideration of the whole operon length (up to 17kb), compatibility and vector carrying capacity, we finally chose the vector pET-28a | ||
+ | <sup>[5]</sup> as backbone. Accordingly, we put the | ||
+ | <i>mamAB</i> | ||
+ | operon into | ||
+ | <i>E.coli</i> | ||
+ | by the vector designed as followed: | ||
</p> | </p> | ||
<div class="project_pic"> | <div class="project_pic"> | ||
<img src="https://static.igem.org/mediawiki/2015/b/bc/CHINA_CD_UESTC_DESIGNmamAB01.png" width="50%"> | <img src="https://static.igem.org/mediawiki/2015/b/bc/CHINA_CD_UESTC_DESIGNmamAB01.png" width="50%"> | ||
− | <p id="pic_illustration"></p> | + | <p id="pic_illustration" style="text-align:center"> |
+ | <strong>Figure 5.</strong> | ||
+ | Schematic of piGEM-AB construction. | ||
+ | </p> | ||
</div> | </div> | ||
+ | |||
<p> | <p> | ||
− | + | Since the length of | |
− | + | <i>mamAB</i> | |
− | + | operon is up to 17kb, it is difficult to directly get its complete gene fragment. After studying the sequence, we divided | |
− | Since the length of <i>mamAB</i> operon is up to 17kb, it is difficult to directly get its complete gene fragment. | + | <i>mamAB</i> |
+ | operon into three parts which amplified from | ||
+ | <strong><i>Magnetospirillum gryphiswaldense MSR-1</i></strong> | ||
+ | , and connected together by the following steps: | ||
</p> | </p> | ||
<div class="project_pic"> | <div class="project_pic"> | ||
<p id="pic_title"></p> | <p id="pic_title"></p> | ||
<img src="https://static.igem.org/mediawiki/2015/8/86/CHINA_CD_UESTC_DESIGNmamAB02.png" width="60%"> | <img src="https://static.igem.org/mediawiki/2015/8/86/CHINA_CD_UESTC_DESIGNmamAB02.png" width="60%"> | ||
− | <p id="pic_illustration"></p> | + | <p id="pic_illustration" style="text-align:center"> |
+ | <strong>Figure 6.</strong> | ||
+ | Schematic of the subclone method. | ||
+ | </p> | ||
</div> | </div> | ||
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<div class="slide" id="slide2" data-slide="4" data-stellar-background-ratio="0.5" style="background-position: 0px 669px;"> | <div class="slide" id="slide2" data-slide="4" data-stellar-background-ratio="0.5" style="background-position: 0px 669px;"> | ||
<div class="container clearfix"> | <div class="container clearfix"> | ||
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<div id="content"> | <div id="content"> | ||
<div class="grid_8"> | <div class="grid_8"> | ||
− | < | + | |
− | + | <h4> | |
+ | 2. | ||
<i>mamGFDC</i> | <i>mamGFDC</i> | ||
− | + | + | |
− | + | ||
− | + | ||
<i>mamXY</i> | <i>mamXY</i> | ||
− | + | + | |
− | + | <i>mms6</i> | |
− | < | + | </h4> |
+ | |||
<p> | <p> | ||
Previous study have shown that although the exact mechanism is not completely understood, these three operons are indispensable in modifying the formation of the magnetosome. Therefore, we built them on one vector to explore its practical effect modification | Previous study have shown that although the exact mechanism is not completely understood, these three operons are indispensable in modifying the formation of the magnetosome. Therefore, we built them on one vector to explore its practical effect modification | ||
− | <sup>[ | + | <sup>[6]</sup> |
. Currently already known as following: | . Currently already known as following: | ||
</p> | </p> | ||
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<li> | <li> | ||
<h5> | <h5> | ||
− | 1 | + | 1) |
− | <strong><i>mamGFDC</i></strong> | + | <strong> |
+ | <i>mamGFDC</i> | ||
+ | </strong> | ||
: | : | ||
</h5> | </h5> | ||
<p> | <p> | ||
Crystal size and shape are mainly regulated by proteins encoded in the | Crystal size and shape are mainly regulated by proteins encoded in the | ||
− | <i> | + | <i>mamGFDC</i> |
operon (composed of the genes | operon (composed of the genes | ||
− | <i>mamC | + | <i>mamC, D, F,</i> |
− | + | and | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
<i>G</i> | <i>G</i> | ||
) and its deletion also leads to a reduction of the size of the magnetite magnetosome crystals | ) and its deletion also leads to a reduction of the size of the magnetite magnetosome crystals | ||
− | <sup>[ | + | <sup>[6,7]</sup> |
+ | . | ||
</p> | </p> | ||
</li> | </li> | ||
<li> | <li> | ||
<h5> | <h5> | ||
− | 2 | + | 2) |
<strong> | <strong> | ||
<i>mamXY</i> | <i>mamXY</i> | ||
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<i>mamXY</i> | <i>mamXY</i> | ||
operon encodes proteins related to the magnetosome membrane ( | operon encodes proteins related to the magnetosome membrane ( | ||
− | <i>mamY | + | <i>mamY, X, Z</i> |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
, and | , and | ||
<i>ftsZ</i> | <i>ftsZ</i> | ||
-like genes) and its deletion causes cells of Magnetospirillum to produce smaller magnetite particles with superparamagnetic characteristics | -like genes) and its deletion causes cells of Magnetospirillum to produce smaller magnetite particles with superparamagnetic characteristics | ||
− | <sup>[ | + | <sup>[8]</sup> |
. | . | ||
</p> | </p> | ||
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<li> | <li> | ||
<h5> | <h5> | ||
− | 3 | + | 3) |
<strong> | <strong> | ||
<i>mms6</i> | <i>mms6</i> | ||
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<i>mms6</i> | <i>mms6</i> | ||
operon contains five genes ( | operon contains five genes ( | ||
− | <i>mms6 | + | <i>mms6, mmsF, mgr4070, mgr4071</i> |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
, and | , and | ||
<i>mgr4074</i> | <i>mgr4074</i> | ||
) | ) | ||
− | <sup>[ | + | <sup>[9]</sup> |
that also appear to be involved in magnetite crystal shape and size. | that also appear to be involved in magnetite crystal shape and size. | ||
</p> | </p> | ||
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</ul> | </ul> | ||
</div> | </div> | ||
+ | |||
<p> | <p> | ||
− | + | We need co-transfer the three vectors into | |
− | < | + | <i>E. coli</i> |
− | and | + | , so the vector we chose be able to co-transformation with the vector pET-28a and pACYCDuet-1 is pCDFDuet-1. The final design of vector is shown in the following figure: |
− | + | ||
− | + | ||
</p> | </p> | ||
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<div class="project_pic"> | <div class="project_pic"> | ||
<img src="https://static.igem.org/mediawiki/2015/a/a1/CHINA_CD_UESTC_DESIGN_GFDC01.png" width="50%"> | <img src="https://static.igem.org/mediawiki/2015/a/a1/CHINA_CD_UESTC_DESIGN_GFDC01.png" width="50%"> | ||
− | <p id="pic_illustration"></p> | + | <p id="pic_illustration" style="text-align:center"> |
+ | <strong>Figure 7.</strong> | ||
+ | Schematic of piGEM-G6X construction. | ||
+ | </p> | ||
</div> | </div> | ||
<P> | <P> | ||
− | + | We decided to get two gene fragments | |
<i>mamXY</i> | <i>mamXY</i> | ||
and | and | ||
− | <i> | + | <i>mamGFDC</i> |
+ | + | ||
<i>mms6</i> | <i>mms6</i> | ||
from | from | ||
− | <i>MSR-1</i> | + | <i><strong>Magnetospirillum gryphiswaldense MSR-1</strong></i> |
− | genome. We respectively designed the method of gene obtain shown in the following figure | + | genome. We respectively designed the method of gene obtain shown in the following figure: |
− | + | ||
− | + | ||
</P> | </P> | ||
<div class="project_pic"> | <div class="project_pic"> | ||
− | <img src="https://static.igem.org/mediawiki/2015/0/00/CHINA_CD_UESTC_DESIGN_GFDC02.png" width="60%"> | + | <img src="https://static.igem.org/mediawiki/2015/0/00/CHINA_CD_UESTC_DESIGN_GFDC02.png" width="60%"> |
− | + | <p id="pic_illustration" style="text-align:center"> | |
− | + | <strong>Figure 8</strong> | |
− | < | + | . Schematic of the piGEM-G6X construction method. |
− | + | </p> | |
− | + | ||
− | + | ||
− | + | </div> | |
− | + | ||
− | + | ||
− | + | ||
− | + | <h4>3. Connection magnetosome with laccase</h4> | |
− | + | <div class="clear"></div> | |
− | + | ||
− | + | ||
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<p> | <p> | ||
− | + | Meanwhile, in order to make laccase enriched, we designed a recombinant vector to fuse express <i>mamW</i> and <i>RFP</i> with the <i>laccase</i>. The protein MamW, a magnetosome transmembrane protein, can connect laccase and magnetosome. The RFP reporter can make the novel structure visible. So we designed the vector piGEM-WRL. As the vector will be co-transferred with another two vectors, we chose the pACYCDuet-1 as the backbone. | |
− | + | ||
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− | + | ||
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− | + | ||
</p> | </p> | ||
<div class="project_pic"> | <div class="project_pic"> | ||
− | <img src="https://static.igem.org/mediawiki/2015/ | + | <img src="https://static.igem.org/mediawiki/2015/9/90/CHINA_CD_UESTC_DESIGN_LACCASE01.png" width="60%"> |
− | <p id="pic_illustration"></p> | + | <p id="pic_illustration"> |
+ | <strong>Figure 9.</strong> Schematic of piGEM-WRL construction. The protein MamW is a magnetosome transmembrane protein<sup>[10]</sup>, <i>mamW</i> gene was amplified from the <strong><i>Magnetospirillum gryphiswaldense MSR-1</i></strong> | ||
+ | </p> | ||
</div> | </div> | ||
+ | <p>In a word, we wanted to fix laccase on magnetosome membrane, and utilize the magnetotaxis of magnetosome to enrich laccase on the cathode. </p> | ||
<div class="reference"> | <div class="reference"> | ||
<h4>Reference</h4> | <h4>Reference</h4> | ||
<p> | <p> | ||
− | [1] | + | [1] Cosnier S, Holzinger M, Goff A L, et al. Recent advances in carbon nanotube-based enzymatic fuel cells.[J]. Frontiers in Bioengineering & Biotechnology, 2014, 2:45. |
</p> | </p> | ||
<p> | <p> | ||
− | [2] Zhang | + | [2] Zhang P. Test method for the laccase activity with ABTS as the substrate[J]. Textile Auxiliaries, 2007. |
</p> | </p> | ||
<p> | <p> | ||
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Latest revision as of 02:31, 19 September 2015
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DESIGN
We mainly designed three vectors respectively carrying mamW + RFP + laccase , mamAB and mamGFDC + mms6 + mamXY . Our purpose is to accomplish our magnetotactic E.coli and fix the laccase on the magnetosome membrane. Finally we put the magnetosomes carrying laccases into our enzymatic bio-fuel cell (EBFC).