Difference between revisions of "Team:CHINA CD UESTC/Design"

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                         <div class="grid_8">
 
                         <div class="grid_8">
 
                             <p>
 
                             <p>
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                                This summer, CHINA_CD_UESTC team made a high-efficiency enzymatic biofuel cell (EBFC) by constructing novel magnetotactic E.coli which can produce laccase.  In our EBFC, laccase is necessary for electrons transfer, we improved previous laccase part (<a href="http://parts.igem.org/Part:BBa_K863005">BBa_K863005</a>) to make it visible . On the one hand, we transferred four operons-- <i>mamAB</i> , <i>mamGFDC</i>, <i>mamXY</i> and <i>mms6</i>, which are related to magnetosomes' formation to make <i>E.coli</i> magnetotactic. On the other hand, we constructed a vector which can express a fusion protein (<i>mamW</i>+<i>RFP</i>+laccase). The protein MamW, a magnetosome transmembrane protein, is a connection between magnetosome and laccase. Therefore, we can immobilize laccase on the magnetosome membrane (MM).
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                            This summer, CHINA_CD_UESTC team made 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 are related to magnetosome formation into <i>E.coli</i>. the modified <i>E.coli<i> can produce laccase-carried magnetosome. Therefore, we can immobilize and enrich laccase on the cathode electrode by magnet. In our project, we improved previous laccase part (<a href="http://parts.igem.org/Part:BBa_K863005">BBa_K863005</a>) and make it visible.<br>  
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The EBFC schematic diagram as following is the final <strong>prototype</strong> of our project:
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                            </p>
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                            <p>
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                              The EBFC schematic diagram as following is our <strong>prototype</strong>
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                                of the project:
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                             </p>
 
                             </p>
 
                             <div class="project_pic">
 
                             <div class="project_pic">
 
                                 <p id="pic_title"></p>
 
                                 <p id="pic_title"></p>
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                                 <img src="https://static.igem.org/mediawiki/2015/f/f7/CHINA_CD_UESTC_DesignOverview.png" width="60%">
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                                 <img src="https://static.igem.org/mediawiki/2015/0/0b/CHINA_CD_UESTC_DESIGN_overview.png" width="60%">
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                                 <p id="pic_illustration">
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                                 <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>
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                                <strong>Figure 1.</strong> Schematic diagram of EBFC. At the anode, glucose is oxidized to gluconolactone, where the electrons are transferred from the GOX to CNT. Catalase decomposes hydrogen peroxide into oxygen and water. At the cathode, electrons are transferred from CNT to laccase where dioxygen is reduced to water.
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                                </p>
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                             </div>
 
                             </div>
  
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                     <div id="content" class="grid_12">
 
                     <div id="content" class="grid_12">
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                         <h3>RFP + Laccase</h3>
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                         <h3>Laccase</h3>
 
                     </div>
 
                     </div>
 
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                     <div class="clear"></div>
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                         <div class="grid_8">
 
                         <div class="grid_8">
 
                             <p>
 
                             <p>
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                                After a review of the relevant literature <sup>[1]</sup>
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                            After a review of the relevant literature<sup>[1]</sup>, 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 laccase. And laccase 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:
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                                , we learned that in the previous bio-fuel cells, the applications of enzyme fuel cell is very wide, and magnetotactic bacteria can generate the magnetosome attracted by magnet. Thereby, we came up with the method that put the laccase into the cell cathode. At the same time, in order to make laccase protein visible we hope to use the reporter gene <i>RFP</i> to locate and mark it. In addition, by changing the environment of laccase, we can find out its optimum environmental conditions using the visualization of RFP. So we designed the vector piGEM-R-Lac.
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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/2015/1/12/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%">
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                                 <p id="pic_illustration"></p>
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                                 <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>
  
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                             <p>
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                             <p>In order to make laccase enriched, we designed a recombinant vector to fuse express <i>mamW</i> and <i>RFP</i> with the laccase. 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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                                <strong>The main role of each gene as follows:</strong>
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                             </p>
 
                             </p>
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                            <div class="list_txt">
 
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                                <ul>
 
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                                    <li>
 
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                                        <p>
 
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                                            (1)
 
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                                            <strong>Laccase:</strong>
 
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                                            Efficient oxidase, catalyzes the substrate to produce electrons, which can be used as a biological cathode in enzyme fuel cell and applied in batteries.
 
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                                        </p>
 
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                                    </li>
 
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                                    <li>
 
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                                        <p>
 
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                                            (2)
 
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                                            <strong>RFP:</strong>
 
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                                            The reporter protein which can locate and content the protein MamW visualized out of the vesicle membrane.
 
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                                        </p>
 
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                                    </li>
 
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                                </ul>
 
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                            </div>
 
  
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                            <p>
 
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                                After constructing this vector completely, we detected whether it work or not by the method of ABTS <sup>[2]</sup>
 
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                                and got the positive result. Furthermore, we learned from literature
 
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                                <sup>[3]</sup>
 
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                                that
 
−
                                <i>mamW</i>
 
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                                gene which located in magnetosome genome had the function of membrane localization.
 
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                                <i>mamW</i>
 
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                                was found in magnetosome  membrane which can help laccase immobilize.
 
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                                <i>MamW</i>
 
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                                is also related to the formation of magnetosome. Therefore, we would like to connect
 
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                                <i>mamW</i>
 
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                                to the working vector. However, the fusion expression of these three proteins may have a difficulty, that no one had studied and done it before, so we designed the following two vectors:
 
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                            </p>
 
 
                             <div class="project_pic">
 
                             <div class="project_pic">
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                                 <p id="pic_title">
+
                                 <p id="pic_title"></p>
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                                    (1)
+
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                                    <i>mamW</i>
+
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                                    +
+
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                                    <i>laccase</i>
+
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                                    : fixed the expressional Laccase on the cell cathode and verified whether MamW protein play a major role in the formation of magnetosome.
+
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                                </p>
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                                <img src="https://static.igem.org/mediawiki/2015/3/3d/CHINA_CD_UESTC_DESIGN_LACCASE04.png" width="60%">
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                                <p id="pic_illustration"></p>
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                            </div>
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+
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                            <div class="project_pic">
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                                <p id="pic_title">
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                                    (2)
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                                    <i>mamW</i>
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                                    +
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                                    <i>RFP</i>
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                                    +
+
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                                    <i>laccase</i>
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                                    : Based on the above vector, RFP protein also can locate and content the MamW protein visualized out of the vesicle membrane, while the contents and expression of Laccase.
+
−
                                </p>
+
 
                                 <img src="https://static.igem.org/mediawiki/2015/9/90/CHINA_CD_UESTC_DESIGN_LACCASE01.png" width="60%">
 
                                 <img src="https://static.igem.org/mediawiki/2015/9/90/CHINA_CD_UESTC_DESIGN_LACCASE01.png" width="60%">
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                                 <p id="pic_illustration"></p>
+
                                 <p id="pic_illustration"><strong>Figure 3<stong>. Schematic of piGEM-WRL construction. The protein MamW is a magnetosome transmembrane protein<sup>[2]</sup>.<i>MamW</i> gene was amplified from the <strong><i>Ms.gryphiswaldense MSR-1</i></strong></p>
 
                             </div>
 
                             </div>
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                             <p>
+
                             <p>After constructing these vectors completely, we detected whether it work or not by the method of ABTS<sup>[3]</sup>.
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                                Wherein,
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                                <i>mamW</i>
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                                gene was amplified from the
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                                <strong><i>Ms.gryphiswaldense MSR-1</i></strong>
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                                . And
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                                <i>laccase</i>
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                                gene was obtained from
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−
                                <a href="http://parts.igem.org/Part:BBa_K863005">BBa_K863005</a>
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                                on the 2015 Kit Plate2. While the
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                                <i>RFP</i>
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                                gene was taken from
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−
                                <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a>
+
−
                                on the 2015 Kit Plate3.
+
 
                             </p>
 
                             </p>
  
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                     <div id="content" class="grid_12">
 
                     <div id="content" class="grid_12">
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                         <h3>Construction of enzymatic biofuel cell (EBFC)</h3>
+
                         <h3>Enzymatic biofuel cell (EBFC)</h3>
 
                     </div>
 
                     </div>
 
                     <div class="clear"></div>
 
                     <div class="clear"></div>
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                     <div id="content">
 
                     <div id="content">
 
                         <div class="grid_8">
 
                         <div class="grid_8">
 +
                        <p>After reading some literatures about EBFC<sup>[4]</sup>, we conceived a common <strong>prototype</strong> of EBFC. </p>
 +
 +
                            <div class="project_pic">
 +
                                <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 4.</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.
 +
                                </p>
 +
                            </div>
 +
 +
 +
 +
 
                             <p>
 
                             <p>
−
                                 As we conceived the prototype of EBFC and read the literature of constructing EBFC
+
                                 The components of the EBFC are listed in the table 1.
−
                                <sup>[4]</sup>
+
−
                                , we prepared materials of components of our Laccase EBFC as following:
+
 
                             </p>
 
                             </p>
 
                             <div class="project_pic">
 
                             <div class="project_pic">
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                             </div>
 
                             </div>
 
                             <p>
 
                             <p>
−
                                We put large and rough surface area carbon paper (Fig. 2A) on both anode and cathode in order to facilitate the attachment of Laccase. The implementation of functional prototype was not just the materials mentioned above, we also bought the Glucose (Fig. 2B,2C), got the Laccase as described above and bought the carbon paper. As we prepared everything already, we successfully constructed a basic EBFC.
+
                              The main materials of our EBFC.  
 
                             </p>
 
                             </p>
 
                             <div class="project_pic">
 
                             <div class="project_pic">
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                                 <img src="https://static.igem.org/mediawiki/2015/3/3b/CHINA_CD_UESTC-DesignPlus02.png" width="70%">
 
                                 <img src="https://static.igem.org/mediawiki/2015/3/3b/CHINA_CD_UESTC-DesignPlus02.png" width="70%">
 
                                 <p id="pic_illustration">
 
                                 <p id="pic_illustration">
−
                                 <strong>Figure 2.</strong> <strong>(A)</strong>Carbon papers on both anode and cathode.<strong>(B)</strong>Glucose enriched on the anode.<strong>(C)</strong>Laccase＋RFP enriched on the cathode.
+
                                 <strong>Figure 5. (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">
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                         <h3>mamAB</h3>
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                         <h3>Magnetosome</h3>
 +
                    </div>
 +
                    <p>In the magnetotactic bacteria, there are four steps to generate magnetosome <sup>[5]</sup> :1-invagination, 2-protein localization, 3-initiation of crystal mineralization, 4-crystal maturation.There exist four operons--<i>mamAB</i>, <i>mamGFDC</i>, <i>mamXY</i> and <i>mms6</i>, which are related to magnetosome formation.</p>
 +
 
 +
 
 +
                    <div id="content" class="grid_12">
 +
                        <h3>1. mamAB</h3>
 
                     </div>
 
                     </div>
 
                     <div class="clear"></div>
 
                     <div class="clear"></div>
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                         <div class="grid_8">
 
                         <div class="grid_8">
 
                             <p>
 
                             <p>
−
                                In the magnetotactic bacteria, there are
+
                             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>[9]</sup>. For consideration of the operon length (up to 17kb), compatibility and vector carrying capacity, we finally chose the backbone vector pET-28a<sup>[6]</sup>. Accordingly, we put the <i>mamAB</i> operon into <i>E.coli</i> by the vector designed as followed:
−
                                <strong>four steps to generate magnetosome</strong>
+
−
                                <sup>[4]</sup>
+
−
                                :1-invagination, 2-protein localization, 3-initiation of crystal mineralization, 4-crystal maturation. Thus, in our project design, we constructed piGEM-AB which is responsible for the formation of magnetosomes.
+
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                             </p>
+
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                            <br>
+
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                            <p>
+
−
                              This section describes the function of the vector <i>piGEM-mamAB</i>. 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>[5]</sup>. Compared to the other three operons which modify the formation of magnetosomes, <i>mamAB</i> can complete its work--producing a basic magnetosome independently. Accordingly, we put the fatal functional operon <i>mamAB</i> 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"><strong>Figure 6.</strong>Schematic of piGEM-AB construction. </p>
 
                             </div>
 
                             </div>
 +
 
                             <p>
 
                             <p>
−
                              For consideration of the operon length (up to 17kb), compatibility and vector carrying capacity, we finally chose the backbone vector <i>pET-28a</i> <sup>[6]</sup>.
+
                             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 <i>mamAB</i> operon into three parts which amplified from <strong><i>Ms.gryphiswaldense MSR-1</i></strong>, and connected together by the following steps:
−
                             </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 <i>mamAB</i> operon into three parts which amplified from MSR-1, 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"><strong>Figure 7.</strong> Schematic of the subclone method.</p>
 
                             </div>
 
                             </div>
−
                            <P>
 
−
                                As we preliminary verified our vector by means of enzyme digestion and sequencing, the results have shown that we have successfully connected the three gene fragments, and the vector was successfully constructed.
 
−
                            </P>
 
−
 
 
                         </div>
 
                         </div>
 
                     </div>
 
                     </div>
Line 294: Line 224:
  
 
                     <div id="content" class="grid_12">
 
                     <div id="content" class="grid_12">
−
                         <h3>
+
                         <h3>2.
−
                             <i>mamGFDC</i>
+
                             <i>mamGFDC</i>+<i>mamXY</i>+<i>mms6</i>
−
                            +
+
−
                            <i>mamXY</i>
+
−
                            +
+
−
                            <i>mms6</i>
+
 
                         </h3>
 
                         </h3>
 
                     </div>
 
                     </div>
Line 306: Line 232:
 
                     <div id="content">
 
                     <div id="content">
 
                         <div class="grid_8">
 
                         <div class="grid_8">
−
                            <p>
 
−
                                In order to further study the formation mechanism of the magnetosome’s shape and size, and control them, we constructed the vector piGEM-G6X which including three important operons of magnetosome:
 
−
                                <i>mamGFDC</i>
 
−
                                ,
 
−
                                <i>mms6</i>
 
−
                                and
 
−
                                <i>mamXY</i>
 
−
                                .
 
−
                            </p>
 
−
                            <br>
 
 
                             <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
Line 330: Line 246:
 
                                         </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>mamGFDC</i> operon (composed of the genes <i>mamC, D, F, </i>and <i>G</i> ) and its deletion also leads to a reduction of the size of the magnetite magnetosome crystals<sup>[7,8]</sup>.
−
                                            <i>mamCD</i>
+
−
                                            operon (composed of the genes
+
−
                                            <i>mamC</i>
+
−
                                            ,
+
−
                                            <i>D</i>
+
−
                                            ,
+
−
                                            <i>F</i>
+
−
                                            , and
+
−
                                            <i>G</i>
+
−
                                            ) and its deletion also leads to a reduction of the size of the magnetite magnetosome crystals
+
−
                                            <sup>[8]</sup>
+
 
                                         </p>
 
                                         </p>
 
                                     </li>
 
                                     </li>
Line 353: Line 258:
 
                                         </h5>
 
                                         </h5>
 
                                         <p>
 
                                         <p>
−
                                             The
+
                                             The <i>mamXY</i> operon encodes proteins related to the magnetosome membrane (<i>mamY, X, Z</i>, and <i>ftsZ</i>-like genes) and its deletion causes cells of Magnetospirillum to produce smaller magnetite particles with superparamagnetic characteristics<sup>[10]</sup> .
−
                                            <i>mamXY</i>
+
−
                                            operon encodes proteins related to the magnetosome membrane (
+
−
                                            <i>mamY</i>
+
−
                                            ,
+
−
                                            <i>X</i>
+
−
                                            ,
+
−
                                            <i>Z</i>
+
−
                                            , and
+
−
                                            <i>ftsZ</i>
+
−
                                            -like genes) and its deletion causes cells of Magnetospirillum to produce smaller magnetite particles with superparamagnetic characteristics
+
−
                                            <sup>[9,10]</sup>
+
−
                                            .
+
 
                                         </p>
 
                                         </p>
 
                                     </li>
 
                                     </li>
Line 377: Line 270:
 
                                         </h5>
 
                                         </h5>
 
                                         <p>
 
                                         <p>
−
                                             The
+
                                             The <i>mms6</i> operon contains five genes (<i>mms6, mmsF, mgr4070, mgr4071</i>, and <i>mgr4074</i>)<sup>[11]</sup>that also appear to be involved in magnetite crystal shape and size.
−
                                            <i>mms6</i>
+
−
                                            operon contains five genes (
+
−
                                            <i>mms6</i>
+
−
                                            ,
+
−
                                            <i>mmsF</i>
+
−
                                            ,
+
−
                                            <i>mgr4070</i>
+
−
                                            ,
+
−
                                            <i>mgr4071</i>
+
−
                                            , and
+
−
                                            <i>mgr4074</i>
+
−
                                            )
+
−
                                            <sup>[11]</sup>
+
−
                                            that also appear to be involved in magnetite crystal shape and size.
+
 
                                         </p>
 
                                         </p>
 
                                     </li>
 
                                     </li>
 
                                 </ul>
 
                                 </ul>
 
                             </div>
 
                             </div>
−
                            <p>
+
 
−
                                In brief, magnetosomes produced by MTB cannot form a chain without those three operons, which will extremely affect its magnetotaxis. So we decided to componentize the genes of this part. And finally we submitted two parts of related genes which are
+
 
−
                                <a href="http://parts.igem.org/Part:BBa_K1779100">BBa_K1779100</a>
+
 
−
                                and
+
                             <p>We need co-transfer the three vectors into <i>E. coli</i>, so the vector we chose be able to co-transformation with the vector pET28a and pACYCDuet-1 is pCDFDuet-1. The final design of vector is shown in the following figure:</p>
−
                                <a href="http://parts.igem.org/Part:BBa_K1779101">BBa_K1779101</a>
+
−
                                .
+
−
                             </p>
+
−
                            <p>
+
−
                                We chose pCDFDuet-1 as our vector, mainly for the following three considerations:
+
−
                            </p>
+
−
                            <div class="list_txt">
+
−
                                <ul>
+
−
                                    <li>
+
−
                                        <h5>
+
−
                                            1.
+
−
                                            <strong>Compatibility</strong>
+
−
                                        </h5>
+
−
                                        <p>
+
−
                                            We need a total of three vectors into
+
−
                                            <i>E. coli</i>
+
−
                                            , so the vector we chose be able to co-transform with the vector pET28a and pACYCDuet-1.
+
−
                                        </p>
+
−
                                    </li>
+
−
                                    <li>
+
−
                                        <h5>
+
−
                                            2.
+
−
                                            <strong>Origin</strong>
+
−
                                        </h5>
+
−
                                        <p>We select the CDF ori as vector's replication origin.</p>
+
−
                                    </li>
+
−
                                    <li>
+
−
                                        <h5>
+
−
                                            3.
+
−
                                            <strong>Carrying Capacity</strong>
+
−
                                        </h5>
+
−
                                        <p>
+
−
                                            Due to the large size of the operon which is 10.4kb, the plasmid must capable to carry this size of gene.
+
−
                                        </p>
+
−
                                    </li>
+
−
                                </ul>
+
−
                            </div>
+
−
                            <h4>The final design of vector is shown in the following figure:</h4>
+
 
                             <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"><strong>Figure 8.</strong> Schematic of piGEM-G6X construction.</p>
 
                             </div>
 
                             </div>
 
                             <P>
 
                             <P>
−
                                 Meanwhile, in order to solve the problem that gene is too large to be directly obtained, we decided to get two gene fragments
+
                                 We decided to get two gene fragments <i>mamXY</i> and <i>GFDC</i> + <i>mms6</i> from <i><strong>Ms.gryphiswaldense MSR-1</strong></i> genome. We respectively designed the method of gene obtain shown in the following figure.  
−
                                <i>mamXY</i>
+
−
                                and
+
−
                                <i>GFDC</i>
+
−
                                +
+
−
                                <i>mms6</i>
+
−
                                from
+
−
                                <i>MSR-1</i>
+
−
                                genome. We respectively designed the method of gene obtain shown in the following figure. The last one,
+
−
                                <i>mamW</i>
+
−
                                was connected on the vector pCDFDuet-1.
+
 
                             </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%"></div>
+
                                 <img src="https://static.igem.org/mediawiki/2015/0/00/CHINA_CD_UESTC_DESIGN_GFDC02.png" width="60%">
−
                            <P>
+
                                 <p id="pic_illustration"><strong>Figure 9</strong>. Schematic of the piGEM-G6X construction method.</p>
−
                                As we preliminary verified our vector by means of enzyme digestion and sequencing, the results have shown that we have successfully connected the two gene fragments, and the vector was successfully constructed.
+
−
                                 <br>
+
−
                                <br></P>
+
−
                        </div>
+
−
                    </div>
+
  
−
                </div>
 
−
            </div>
 
−
            <div class="slide" id="slide2" data-slide="4" data-stellar-background-ratio="0.5" style="background-position: 0px 669px;">
 
−
                <div class="container clearfix">
 
  
−
                    <div id="content" class="grid_12">
+
                            </div>
−
                        <h3>The promoter verification</h3>
+
 
−
                    </div>
+
                            <h3>3. The magnetosome verification</h3>
 
                     <div class="clear"></div>
 
                     <div class="clear"></div>
−
 
−
                    <div id="content">
 
−
                        <div class="grid_8">
 
 
                             <p>
 
                             <p>
−
                                In order to find the reason why the magnetosome was not formed in the
+
                            In order to verify the magnetosome was formed in the <i>E.coli</i>, we constructed 4 vectors to investigate the operons’ promoters. We chose pSB1C3 as backbone, and replaced the PlacI of the part <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a>. In order to further study the formation mechanism of the magnetosome, we construct several vectors to investigate every gene of the operons.
−
                                <i>E.coli</i>
+
−
                                , we constructed several vectors to investigate the operons’ promoters. We chose pSB1C3 as backbone, and replaced the
+
−
                                <i>PlacI</i>
+
−
                                of the part
+
−
                                <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a>
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                                or replaced
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                                <i>RFP</i>
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                                which was the first genes of every operons.
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                                 <img src="https://static.igem.org/mediawiki/2015/e/e6/CHINA_CD_UESTC-DesignPlus05.png" width="50%">
 
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                                 <p id="pic_illustration"><strong>Figure 10.</strong> Schematic of the verified vectors construction.</p>
 
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                                     [2] Zhang Peng (2007). “Test method for the Laccase activity with ABTS as the substrate.” China Academic Journal Electronic Publishing House 24:1
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                                     [2] Isabel Kolinko, Anna Lohße, Sarah Borg, et al. (2014). “Biosynthesis of magnetic nanostructures in a foreign organism by transfer of bacterial magnetosome gene clusters.” Nature Nanotechnology 9: 193-197, doi:10.1038/nnano.2014.13
 
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                                     [3] Isabel Kolinko, Anna Lohße, Sarah Borg, et al. (2014). “Biosynthesis of magnetic nanostructures in a foreign organism by transfer of bacterial magnetosome gene clusters.” Nature Nanotechnology 9: 193-197, doi:10.1038/nnano.2014.13
+
                                     [3] Zhang Peng (2007). “Test method for the Laccase activity with ABTS as the substrate.” China Academic Journal Electronic Publishing House 24:1
 
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                                     [4] Ana Carolina V. Araujo 1, Fernanda Abreu 1, Karen Tavares Silva 1,2, Dennis A. Bazylinski 3 and Ulysses Lins 1,* Magnetotactic Bacteria as Potential Sources of Bioproducts. Mar. Drugs 2015, 13, 389-430; doi:10.3390/md13010389
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                                     [4] Abdelkader Zebda1,2, Chantal Gondran1, Alan Le Goff1. Mediatorless high-power glucose biofuel cells based on compressed carbon nanotube-enzyme electrodes nature communications | 2:370 | DOI: 10.1038/ncomms1365
 
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Revision as of 17:25, 16 September 2015

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DESIGN

  We mainly designed three vectors respectively carrying mamW+RFP+laccase , mamAB and mamGFDC + mamXY+ mms6. 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).

Overview

This summer, CHINA_CD_UESTC team made a high-efficiency enzymatic biofuel cell (EBFC) by enriching the laccase on the cathode electrode. we transferred four operons-- mamAB , mamGFDC, mamXY and mms6, which are related to magnetosome formation into E.coli. the modified E.coli can produce laccase-carried magnetosome. Therefore, we can immobilize and enrich laccase on the cathode electrode by magnet. In our project, we improved previous laccase part (BBa_K863005) and make it visible.
The EBFC schematic diagram as following is the final prototype of our project:

Figure 1. 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.

Laccase

After a review of the relevant literature[1], 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 RFP with the laccase. And laccase gene was obtained from BBa_K863005 on the 2015 Kit Plate2. While the RFP gene was taken from BBa_E1010 on the 2015 Kit Plate3. We designed the vector piGEM-RL as below:

Figure 2.Schematic of piGEM-RL construction. Laccase: efficient oxidase, catalyzes the substrate to produce electrons and environmentally friendly. RFP: the reporter protein.

In order to make laccase enriched, we designed a recombinant vector to fuse express mamW and RFP with the laccase. 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.

Figure 3. Schematic of piGEM-WRL construction. The protein MamW is a magnetosome transmembrane protein[2].MamW gene was amplified from the Ms.gryphiswaldense MSR-1

After constructing these vectors completely, we detected whether it work or not by the method of ABTS[3].

Enzymatic biofuel cell (EBFC)

After reading some literatures about EBFC[4], we conceived a common prototype of EBFC.

Figure 4. 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.

The components of the EBFC are listed in the table 1.

The main materials of our EBFC.

Figure 5. (A) Carbon papers as the electrode.(B) Glucose oxidase on the anode.(C) RFP＋laccase on the cathode.

Magnetosome

In the magnetotactic bacteria, there are four steps to generate magnetosome [5] :1-invagination, 2-protein localization, 3-initiation of crystal mineralization, 4-crystal maturation.There exist four operons--mamAB, mamGFDC, mamXY and mms6, which are related to magnetosome formation.

1. mamAB

This section describes the function of the vector piGEM-mamAB. It carries mamAB operon whose length is up to 17kb. Previous studies have shown that mamAB operon is one of the four core operons related to magnetosomes formation[9]. For consideration of the operon length (up to 17kb), compatibility and vector carrying capacity, we finally chose the backbone vector pET-28a[6]. Accordingly, we put the mamAB operon into E.coli by the vector designed as followed:

Figure 6.Schematic of piGEM-AB construction.

Since the length of mamAB operon is up to 17kb, it is difficult to directly get its complete gene fragment. After studying the sequence, we divided mamAB operon into three parts which amplified from Ms.gryphiswaldense MSR-1, and connected together by the following steps:

Figure 7. Schematic of the subclone method.

2. mamGFDC+mamXY+mms6

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 [7] . Currently already known as following:

  • 1. mamGFDC ：

    Crystal size and shape are mainly regulated by proteins encoded in the mamGFDC operon (composed of the genes mamC, D, F, and G ) and its deletion also leads to a reduction of the size of the magnetite magnetosome crystals[7,8].

  • 2. mamXY ：

    The mamXY operon encodes proteins related to the magnetosome membrane (mamY, X, Z, and ftsZ-like genes) and its deletion causes cells of Magnetospirillum to produce smaller magnetite particles with superparamagnetic characteristics[10] .

  • 3. mms6 ：

    The mms6 operon contains five genes (mms6, mmsF, mgr4070, mgr4071, and mgr4074)[11]that also appear to be involved in magnetite crystal shape and size.

We need co-transfer the three vectors into E. coli, so the vector we chose be able to co-transformation with the vector pET28a and pACYCDuet-1 is pCDFDuet-1. The final design of vector is shown in the following figure:

Figure 8. Schematic of piGEM-G6X construction.

We decided to get two gene fragments mamXY and GFDC + mms6 from Ms.gryphiswaldense MSR-1 genome. We respectively designed the method of gene obtain shown in the following figure.

Figure 9. Schematic of the piGEM-G6X construction method.

3. The magnetosome verification

In order to verify the magnetosome was formed in the E.coli, we constructed 4 vectors to investigate the operons’ promoters. We chose pSB1C3 as backbone, and replaced the PlacI of the part BBa_J04450. In order to further study the formation mechanism of the magnetosome, we construct several vectors to investigate every gene of the operons.

Figure 10. Schematic of the verified vectors construction.

Reference

[1] Serge Cosnier, Michael Holzinger, Alan Le Goff (2014). “Recent advances in carbon nanotube-based enzymatic fuel cells.” Bioengineering and Biotechnology 2:45, doi: 10.3389/fbioe.2014.00045

[2] Isabel Kolinko, Anna Lohße, Sarah Borg, et al. (2014). “Biosynthesis of magnetic nanostructures in a foreign organism by transfer of bacterial magnetosome gene clusters.” Nature Nanotechnology 9: 193-197, doi:10.1038/nnano.2014.13

[3] Zhang Peng (2007). “Test method for the Laccase activity with ABTS as the substrate.” China Academic Journal Electronic Publishing House 24:1

[4] Abdelkader Zebda1,2, Chantal Gondran1, Alan Le Goff1. Mediatorless high-power glucose biofuel cells based on compressed carbon nanotube-enzyme electrodes nature communications | 2:370 | DOI: 10.1038/ncomms1365

[5] Anna Lohße1, Susanne Ullrich1, Emanuel Katzmann1, Sarah Borg1, Gerd Wanner1, Michael Richter2,Birgit Voigt3, Thomas Schweder4, Dirk Schu¨ ler1*.Functional Analysis of the Magnetosome Island in Magnetospirillum gryphiswaldense: The mamAB Operon Is Sufficient for Magnetite Biomineralization

[6] Citation: Lee HY, Khosla C (2007) Bioassay-guided evolution of glycosylated macrolide antibiotics in Escherichia coli. PLoS Biol 5(2): e45. doi:10.1371/journal.pbio.0050045

[7] Ana Carolina V. Araujo; Fernanda Abreu; Karen Tavares Silva; Dennis A. Bazylinski; Ulysses Lins. Magnetotactic Bacteria as Potential Sources of Bioproducts.Mar. Drugs 2015,13,389-430

[8] Scheffel, A.; Gärdes, A.; Grünberg, K.; Wanner, G.; Schüler, D. The major magnetosome proteins MamGFDC are not essential for magnetite biomineralization in Magnetospirillum gryphiswaldense but regulate the size of magnetosome crystals. J. Bacteriol. 2008, 190, 377–386

[9] Lohße, A.; Ullrich, S.; Katzmann, E.; Borg, S.; Wanner, G.; Richter, M.; Voigt, B.; Schweder, T.; Schüler, D. Functional analysis of the magnetosome island in Magnetospirillum gryphiswaldense: The mamAB operon is sufficient for magnetite biomineralization. PLoS One 2011, 6, doi:10.1371/journal.pone.0025561

[10] Ding, Y.; Li, J.; Liu, J.; Yang, J.; Jiang, W.; Tian, J.; Li, Y.; Pan, Y.; Li, J. Deletion of the ftsZ-like gene results in the production of superparamagnetic magnetite magnetosomes in Magnetospirillum gryphiswaldense. J. Bacteriol. 2010, 192, 1097–1105

[11] Murat, D.; Quinlan, A.; Vali, H.; Komeili, A. Comprehensive genetic dissection of the magnetosome gene island reveals the step-wise assembly of a prokaryotic organelle. Proc. Natl. Acad. Sci. USA 2010, 107, 5593–5598