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

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                <B>DESIGN</B>
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<B>DESIGN</B>
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                &nbsp;&nbsp;We mainly designed three vectors respectively carrying <i>mamW</i>+<i>RFP</i>+laccase , <i>mamAB</i> and <i>mamGFDC</i> + <i>mamXY</i>+ <i>mms6</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).  
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&nbsp;&nbsp;We mainly designed three vectors respectively carrying <i>mamW</i>
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+ <i>RFP</i>
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        </div>
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+laccase ,
 +
<i>mamAB</i>
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and
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<i>mamGFDC</i>
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 +
<i>mamXY</i>
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+
 +
<i>mms6</i>
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. Our purpose is to accomplish our magnetotactic
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<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).
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</p>
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                        <h3>Overview</h3>
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<h3>Overview</h3>
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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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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--
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The EBFC schematic diagram as following is the final <strong>prototype</strong> of our project:
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<i>mamAB</i>
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                            </p>
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,
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                            <div class="project_pic">
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<i>mamGFDC</i>
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                                <p id="pic_title"></p>
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,
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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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<i>mamXY</i>
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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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and
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                            </div>
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<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.</p>
 +
<P>
 +
The EBFC schematic diagram as following is the final <strong>prototype</strong>
 +
of our project:</P>
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<div class="project_pic">
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<p id="pic_title"></p>
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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"> <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.
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</p>
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<div id="content" class="grid_12">
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                        <h3>Laccase</h3>
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<h3>Laccase</h3>
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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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After a review of the relevant literature <sup>[1]</sup>
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                            </p>
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, 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
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                            <div class="project_pic">
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<i>RFP</i>
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                                <p id="pic_title"></p>
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with the laccase. And laccase gene was obtained from
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                                <img src="https://static.igem.org/mediawiki/2015/1/12/CHINA_CD_UESTC_DESIGN_LACCASE02.png" width="60%">
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<a href="http://parts.igem.org/Part:BBa_K863005">BBa_K863005</a>
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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>
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on the 2015 Kit Plate2. While the
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                            </div>
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<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:
 +
</p>
 +
<div class="project_pic">
 +
<p id="pic_title"></p>
 +
<img src="https://static.igem.org/mediawiki/2015/1/12/CHINA_CD_UESTC_DESIGN_LACCASE02.png" width="60%">
 +
<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>
  
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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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<p>
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                            </p>
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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.
 +
</p>
  
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                            <div class="project_pic">
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<div class="project_pic">
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                                <p id="pic_title"></p>
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<p id="pic_title"></p>
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                                <img src="https://static.igem.org/mediawiki/2015/9/90/CHINA_CD_UESTC_DESIGN_LACCASE01.png" width="60%">
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<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"><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>
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<p id="pic_illustration">
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                            </div>
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<strong>Figure 3
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                            <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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<stong>
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                            </p>
+
. 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>
 +
<p>
 +
After constructing these vectors completely, we detected whether it work or not by the method of ABTS
 +
<sup>[3]</sup>
 +
.
 +
</p>
  
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<div id="content" class="grid_12">
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                        <h3>Enzymatic biofuel cell (EBFC)</h3>
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<h3>Enzymatic biofuel cell (EBFC)</h3>
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                        <p>After reading some literatures about EBFC<sup>[4]</sup>, we conceived a common <strong>prototype</strong> of EBFC. </p>
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<p>
 +
After reading some literatures about EBFC
 +
<sup>[4]</sup>
 +
, we conceived a common
 +
<strong>prototype</strong>
 +
of EBFC.
 +
</p>
  
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                            <div class="project_pic">
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<div class="project_pic">
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                                <p id="pic_title"></p>
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<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/f/f7/CHINA_CD_UESTC_DesignOverview.png" width="60%">
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                                <p id="pic_illustration">
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<p id="pic_illustration">
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                                <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.
+
<strong>Figure 4.</strong>
−
                                </p>
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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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                            </div>
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</p>
 +
</div>
  
 +
<p>The components of the EBFC are listed in the table 1.</p>
 +
<div class="project_pic">
 +
<p id="pic_title"></p>
 +
<img src="https://static.igem.org/mediawiki/2015/a/a8/CHINA_CD_UESTC-DesignPlus01.png" width="60%">
 +
<p id="pic_illustration"></p>
 +
</div>
 +
<p>The main materials of our EBFC.</p>
 +
<div class="project_pic">
 +
<p id="pic_title"></p>
 +
<img src="https://static.igem.org/mediawiki/2015/3/3b/CHINA_CD_UESTC-DesignPlus02.png" width="70%">
 +
<p id="pic_illustration">
 +
<strong>Figure 5. (A)</strong>
 +
Carbon papers as the electrode.
 +
<strong>(B)</strong>
 +
Glucose oxidase on the anode.
 +
<strong>(C)</strong>
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RFP＋laccase on the cathode.
 +
</p>
 +
</div>
  
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</div>
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</div>
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</div>
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                                The components of the EBFC are listed in the table 1.
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<h3>Magnetosome</h3>
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                            </p>
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</div>
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                            <div class="project_pic">
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<p>
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                                <p id="pic_title"></p>
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In the magnetotactic bacteria, there are four steps to generate magnetosome
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                                <img src="https://static.igem.org/mediawiki/2015/a/a8/CHINA_CD_UESTC-DesignPlus01.png" width="60%">
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<sup>[5]</sup>
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                                <p id="pic_illustration"></p>
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:1-invagination, 2-protein localization, 3-initiation of crystal mineralization, 4-crystal maturation.There exist four operons--
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                            </div>
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<i>mamAB</i>
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                            <p>
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,
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                              The main materials of our EBFC.
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<i>mamGFDC</i>
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                            </p>
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,
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                            <div class="project_pic">
+
<i>mamXY</i>
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                                <p id="pic_title"></p>
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and
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                                <img src="https://static.igem.org/mediawiki/2015/3/3b/CHINA_CD_UESTC-DesignPlus02.png" width="70%">
+
<i>mms6</i>
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                                <p id="pic_illustration">
+
, which are related to magnetosome formation.
−
                                <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.
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</p>
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<div id="content" class="grid_12">
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<h3>1. mamAB</h3>
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<p>
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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:
 +
</p>
 +
<div class="project_pic">
 +
<img src="https://static.igem.org/mediawiki/2015/b/bc/CHINA_CD_UESTC_DESIGNmamAB01.png" width="50%">
 +
<p id="pic_illustration">
 +
<strong>Figure 6.</strong>
 +
Schematic of piGEM-AB construction.
 +
</p>
 +
</div>
  
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                    <div id="content" class="grid_12">
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<p>
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                        <h3>Magnetosome</h3>
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Since the length of
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                    </div>
+
<i>mamAB</i>
−
                    <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>
+
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>
 +
<div class="project_pic">
 +
<p id="pic_title"></p>
 +
<img src="https://static.igem.org/mediawiki/2015/8/86/CHINA_CD_UESTC_DESIGNmamAB02.png" width="60%">
 +
<p id="pic_illustration">
 +
<strong>Figure 7.</strong>
 +
Schematic of the subclone method.
 +
</p>
 +
</div>
 +
</div>
 +
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<h3>
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2.
−
                            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:
+
<i>mamGFDC</i>
−
                            </p>
+
+
−
                            <div class="project_pic">
+
<i>mamXY</i>
−
                                <img src="https://static.igem.org/mediawiki/2015/b/bc/CHINA_CD_UESTC_DESIGNmamAB01.png" width="50%">
+
+
−
                                <p id="pic_illustration"><strong>Figure 6.</strong>Schematic of piGEM-AB construction. </p>
+
<i>mms6</i>
−
                            </div>
+
</h3>
 +
</div>
 +
<div class="clear"></div>
  
−
                            <p>
+
<div id="content">
−
                            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:
+
<div class="grid_8">
−
                            </p>
+
<p>
−
                            <div class="project_pic">
+
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
−
                                <p id="pic_title"></p>
+
<sup>[7]</sup>
−
                                <img src="https://static.igem.org/mediawiki/2015/8/86/CHINA_CD_UESTC_DESIGNmamAB02.png" width="60%">
+
. Currently already known as following:
−
                                <p id="pic_illustration"><strong>Figure 7.</strong> Schematic of the subclone method.</p>
+
</p>
−
                            </div>
+
<div class="list_txt">
−
                        </div>
+
<ul>
−
                    </div>
+
<li>
 +
<h5>
 +
1.
 +
<strong>
 +
<i>mamGFDC</i>
 +
</strong>
 +
：
 +
</h5>
 +
<p>
 +
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>
 +
.
 +
</p>
 +
</li>
 +
<li>
 +
<h5>
 +
2.
 +
<strong>
 +
<i>mamXY</i>
 +
</strong>
 +
：
 +
</h5>
 +
<p>
 +
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>
 +
.
 +
</p>
 +
</li>
 +
<li>
 +
<h5>
 +
3.
 +
<strong>
 +
<i>mms6</i>
 +
</strong>
 +
：
 +
</h5>
 +
<p>
 +
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.
 +
</p>
 +
</li>
 +
</ul>
 +
</div>
  
−
                </div>
+
<p>
−
            </div>
+
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>
 +
<div class="project_pic">
 +
<img src="https://static.igem.org/mediawiki/2015/a/a1/CHINA_CD_UESTC_DESIGN_GFDC01.png" width="50%">
 +
<p id="pic_illustration">
 +
<strong>Figure 8.</strong>
 +
Schematic of piGEM-G6X construction.
 +
</p>
 +
</div>
 +
<P>
 +
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.
 +
</P>
 +
<div class="project_pic">
 +
<img src="https://static.igem.org/mediawiki/2015/0/00/CHINA_CD_UESTC_DESIGN_GFDC02.png" width="60%">
 +
<p id="pic_illustration">
 +
<strong>Figure 9</strong>
 +
. Schematic of the piGEM-G6X construction method.
 +
</p>
  
−
            <div class="slide" id="slide2" data-slide="4" data-stellar-background-ratio="0.5" style="background-position: 0px 669px;">
+
</div>
−
                <div class="container clearfix">
+
  
−
                    <div id="content" class="grid_12">
+
<h3>3. The magnetosome verification</h3>
−
                        <h3>2.
+
<div class="clear"></div>
−
                            <i>mamGFDC</i>+<i>mamXY</i>+<i>mms6</i>
+
<p>
−
                        </h3>
+
In order to verify the magnetosome was formed in the
−
                    </div>
+
<i>E.coli</i>
−
                    <div class="clear"></div>
+
, 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.
 +
</p>
 +
<div class="project_pic">
 +
<img src="https://static.igem.org/mediawiki/2015/e/e6/CHINA_CD_UESTC-DesignPlus05.png" width="50%">
 +
<p id="pic_illustration">
 +
<strong>Figure 10.</strong>
 +
Schematic of the verified vectors construction.
 +
</p>
 +
</div>
 +
<div class="reference">
 +
<h4>Reference</h4>
 +
<p>
 +
[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
 +
</p>
 +
<p>
 +
[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
 +
</p>
 +
<p>
 +
[3] Zhang Peng (2007). “Test method for the Laccase activity with ABTS as the substrate.” China Academic Journal Electronic Publishing House 24:1
 +
</p>
 +
<p>
 +
[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
 +
</p>
 +
<p>
 +
[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
 +
</p>
 +
<p>
 +
[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
 +
</p>
 +
<p>
 +
[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
 +
</p>
 +
<p>
 +
[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
 +
</p>
 +
<p>
 +
[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
 +
</p>
  
−
                    <div id="content">
+
<p>
−
                        <div class="grid_8">
+
[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
−
                            <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
+
<p>
−
                                <sup>[7]</sup>
+
[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
−
                                . Currently already known as following:
+
</p>
−
                            </p>
+
−
                            <div class="list_txt">
+
−
                                <ul>
+
−
                                    <li>
+
−
                                        <h5>
+
−
                                            1.
+
−
                                            <strong><i>mamGFDC</i></strong>
+
−
                                            ：
+
−
                                        </h5>
+
−
                                        <p>
+
−
                                            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>.
+
−
                                        </p>
+
−
                                    </li>
+
−
                                    <li>
+
−
                                        <h5>
+
−
                                            2.
+
−
                                            <strong>
+
−
                                                <i>mamXY</i>
+
−
                                            </strong>
+
−
                                            ：
+
−
                                        </h5>
+
−
                                        <p>
+
−
                                            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> .
+
−
                                        </p>
+
−
                                    </li>
+
−
                                    <li>
+
−
                                        <h5>
+
−
                                            3.
+
−
                                            <strong>
+
−
                                                <i>mms6</i>
+
−
                                            </strong>
+
−
                                            ：
+
−
                                        </h5>
+
−
                                        <p>
+
−
                                            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.
+
−
                                        </p>
+
−
                                    </li>
+
−
                                </ul>
+
−
                            </div>
+
  
 +
</div>
  
 +
</div>
 +
</div>
  
−
                            <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>
+
</div>
−
                            <div class="project_pic">
+
</div>
−
                                <img src="https://static.igem.org/mediawiki/2015/a/a1/CHINA_CD_UESTC_DESIGN_GFDC01.png" width="50%">
+
</div>
−
                                <p id="pic_illustration"><strong>Figure 8.</strong> Schematic of piGEM-G6X construction.</p>
+
</div>
−
                            </div>
+
−
                            <P>
+
−
                                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.
+
−
                            </P>
+
−
                            <div class="project_pic">
+
−
                                <img src="https://static.igem.org/mediawiki/2015/0/00/CHINA_CD_UESTC_DESIGN_GFDC02.png" width="60%">
+
−
                                <p id="pic_illustration"><strong>Figure 9</strong>. Schematic of the piGEM-G6X construction method.</p>
+
−
 
+
−
 
+
−
                            </div>
+
−
 
+
−
                            <h3>3. The magnetosome verification</h3>
+
−
                    <div class="clear"></div>
+
−
                            <p>
+
−
                            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.
+
−
                            </p>
+
−
                            <div class="project_pic">
+
−
                                <img src="https://static.igem.org/mediawiki/2015/e/e6/CHINA_CD_UESTC-DesignPlus05.png" width="50%">
+
−
                                <p id="pic_illustration"><strong>Figure 10.</strong> Schematic of the verified vectors construction.</p>
+
−
                            </div>
+
−
                            <div class="reference">
+
−
                                <h4>Reference</h4>
+
−
                                <p>
+
−
                                    [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
+
−
                                </p>
+
−
                                <p>
+
−
                                    [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
+
−
                                </p>
+
−
                                <p>
+
−
                                    [3] Zhang Peng (2007). “Test method for the Laccase activity with ABTS as the substrate.” China Academic Journal Electronic Publishing House 24:1
+
−
                                </p>
+
−
                                <p>
+
−
                                    [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
+
−
                                </p>
+
−
                                <p>
+
−
                                    [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
+
−
                                </p>
+
−
                                <p>
+
−
                                    [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
+
−
                                </p>
+
−
                                <p>
+
−
                                    [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
+
−
                                </p>
+
−
                                <p>
+
−
                                    [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
+
−
                                </p>
+
−
                                <p>
+
−
                                    [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
+
−
                                </p>
+
−
 
+
−
                                <p>
+
−
                                    [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
+
−
                                </p>
+
−
                                <p>
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Revision as of 17:27, 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.colican 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