Difference between revisions of "Team:CHINA CD UESTC/Results"
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<p class="blockWords"> | <p class="blockWords"> | ||
− | | + | Finally, we got the fusion proteins MamW + RFP + laccase and RFP + laccase. The fusion protein RFP + laccase worked very well in our EBFC. What's more, after we co-transferred the two vectors piGEM-AB and piGEM-G6X into <i>Escherichia coli BL21(DE3)</i>, there were something interesting appeared in the bacteria! |
</p> | </p> | ||
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<div id="content"> | <div id="content"> | ||
<div class="grid_8"> | <div class="grid_8"> | ||
− | <h2> | + | <h2 style="font-size:30px">Vectors construction and activity detection of laccase</h2> |
<div class="project_pic"> | <div class="project_pic"> | ||
<p id="pic_title"> | <p id="pic_title"> | ||
− | <strong>1. Amplification of target genes.</strong> We respectively amplified <i>mamW</i>, <i>RFP</i> and <i>laccase</i> by common PCR (Fig. 1A). In order to make | + | <strong>1. Amplification of target genes.</strong> We respectively amplified <i>mamW</i>, <i>RFP</i> and <i>laccase</i> by common PCR (Fig. 1A). In order to make laccase visible, we combined <i>RFP</i> with <i>laccase</i>. In order to immobilize laccase, we combined <i>mamW+RFP+laccase</i> and <i>RFP</i>+<i>laccase</i> by fusion PCR (Fig. 1B).<br> |
</p> | </p> | ||
<img src="https://static.igem.org/mediawiki/2015/f/f8/CHINA_CD_UESTC_RESULTfin01.png" width="60%"> | <img src="https://static.igem.org/mediawiki/2015/f/f8/CHINA_CD_UESTC_RESULTfin01.png" width="60%"> | ||
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</p> | </p> | ||
</div> | </div> | ||
+ | <br> | ||
<div class="project_pic"> | <div class="project_pic"> | ||
<p id="pic_title"> | <p id="pic_title"> | ||
− | <strong>2. Verification of vectors.</strong> We purified the PCR products (Fig. 1B) and successfully inserted the fragments into pACYCDuet-1, and named piGEM-WRL and piGEM-RL respectively. We verified them using digestion (Fig. 2) and sequencing. | + | <strong>2. Verification of vectors.</strong> We purified the PCR products (Fig. 1B) and successfully inserted the fragments into pACYCDuet-1, and named piGEM-WRL and piGEM-RL respectively. We verified them using digestion (Fig. 2) and sequencing.<br> |
</p> | </p> | ||
<img src="https://static.igem.org/mediawiki/2015/6/66/CHINA_CD_UESTC_RESULTfin02.png" width="60%"> | <img src="https://static.igem.org/mediawiki/2015/6/66/CHINA_CD_UESTC_RESULTfin02.png" width="60%"> | ||
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<div class="project_pic"> | <div class="project_pic"> | ||
<p id="pic_title"> | <p id="pic_title"> | ||
− | <strong>3. Transformation and inducible expression.</strong> We transformed the piGEM-RL into <i>BL21(DE3)</i> and conducted inducible expression. The color (Fig. 3A) and concentration (Fig. 3B) of bacterium liquid are as followed: | + | <strong>3. Transformation and inducible expression of RFP+laccase.</strong> We transformed the piGEM-RL into <i>BL21(DE3)</i> and conducted inducible expression. The color (Fig. 3A) and concentration (Fig. 3B) of bacterium liquid are as followed:<br> |
</p> | </p> | ||
<img src="https://static.igem.org/mediawiki/2015/7/7e/CHINA_CD_UESTC_RESULTfin03.png" width="70%"> | <img src="https://static.igem.org/mediawiki/2015/7/7e/CHINA_CD_UESTC_RESULTfin03.png" width="70%"> | ||
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</div> | </div> | ||
<div class="project_pic"> | <div class="project_pic"> | ||
− | <p id="pic_title"><strong>4. Test the expression of RFP+ | + | <p id="pic_title"><strong>4. Test the expression of RFP+laccase.</strong> We broke the cells in boiling water for 10min and run SDS-PAGE with this sample (Fig. 4).</p><br> |
<img src="https://static.igem.org/mediawiki/2015/6/6c/CHINA_CD_UESTC_RESULTfin04.png" width="30%"> | <img src="https://static.igem.org/mediawiki/2015/6/6c/CHINA_CD_UESTC_RESULTfin04.png" width="30%"> | ||
<p id="pic_illustration"> | <p id="pic_illustration"> | ||
− | <strong>Figure 4.</strong> Testing expression of RFP+ | + | <strong>Figure 4.</strong> Testing expression of RFP+laccase in <i>E.coli.</i> M: marker. Lane 1, bacteria untransformed. Lane 2, Bacteria which contain piGEM-RL. Induced at 37centigrades, 180rpm, 10 hours with 0.5mM IPTG. |
</p> | </p> | ||
</div> | </div> | ||
<p> | <p> | ||
− | In the gel we found that the band located <strong>near 84KDa is RFP+ | + | In the gel we found that the band located <strong>near 84KDa is RFP+laccase.</strong><br><br> |
</p> | </p> | ||
<div class="project_pic"> | <div class="project_pic"> | ||
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The higher concentration of laccase showed redder. | The higher concentration of laccase showed redder. | ||
<strong>(B)</strong> | <strong>(B)</strong> | ||
− | The activity of RFP+ | + | The activity of RFP+laccase. Ultrasonic Cell Disruptor to crush the bacterium in ice-bath. Collect the Supernatant and detect the activity of laccase by ABTS method. The 1mL supernate equal to the 5mL bacterium liquid which were cultivated for different times. |
</p> | </p> | ||
</div> | </div> | ||
<div class="project_pic"> | <div class="project_pic"> | ||
<p id="pic_title"> | <p id="pic_title"> | ||
− | <strong>6. Transformation and inducible expression.</strong> We transformed piGEM-WRL into <i>BL21(DE3)</i> and conducted inducible expression. We got a series of samples which was cultivated at different times and chose one to compare with <i>BL21(DE3)</i> untransformed (Fig. 6A). Then, we detected the activity of laccase (Fig. 6B). | + | <strong>6. Transformation and inducible expression of mamW+RFP+laccase.</strong> We transformed piGEM-WRL into <i>BL21(DE3)</i> and conducted inducible expression. We got a series of samples which was cultivated at different times and chose one to compare with <i>BL21(DE3)</i> untransformed (Fig. 6A). Then, we detected the activity of laccase (Fig. 6B). |
</p> | </p> | ||
<img src="https://static.igem.org/mediawiki/2015/8/8b/CHINA_CD_UESTC_RESULTfin06.png" width="70%"> | <img src="https://static.igem.org/mediawiki/2015/8/8b/CHINA_CD_UESTC_RESULTfin06.png" width="70%"> | ||
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The left is <i>BL21(DE3)</i> untransformed and the right is <i>BL21(DE3)</i> transformed with piGEM-WRL. | The left is <i>BL21(DE3)</i> untransformed and the right is <i>BL21(DE3)</i> transformed with piGEM-WRL. | ||
<strong>(B)</strong> | <strong>(B)</strong> | ||
− | The activity of MamW + RFP + | + | The activity of MamW + RFP + laccase. |
</p> | </p> | ||
− | </div> | + | </div><br> |
<div class="project_pic"> | <div class="project_pic"> | ||
<p id="pic_title"><strong>7. The activity comparation of two laccases.</strong> We compared the two enzyme activity curves(Fig. 7).</p> | <p id="pic_title"><strong>7. The activity comparation of two laccases.</strong> We compared the two enzyme activity curves(Fig. 7).</p> | ||
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<p id="pic_title"><strong>1. The sample EBFC 1.0.</strong> We made this device using discarded bottles with the proton exchange membrane in the middle (Fig. 8A). Fix the EBFC 1.0 in the foam board, add each component (10 ml in total) into the device according to the <a href="https://2015.igem.org/Team:CHINA_CD_UESTC/Protocol">protocol</a>. Use multimeter and oscilloscope to test voltage (Fig. 8B).</p> | <p id="pic_title"><strong>1. The sample EBFC 1.0.</strong> We made this device using discarded bottles with the proton exchange membrane in the middle (Fig. 8A). Fix the EBFC 1.0 in the foam board, add each component (10 ml in total) into the device according to the <a href="https://2015.igem.org/Team:CHINA_CD_UESTC/Protocol">protocol</a>. Use multimeter and oscilloscope to test voltage (Fig. 8B).</p> | ||
<img src="https://static.igem.org/mediawiki/2015/9/99/CHINA_CD_UESTC_RESULTfin08.png" width="60%"> | <img src="https://static.igem.org/mediawiki/2015/9/99/CHINA_CD_UESTC_RESULTfin08.png" width="60%"> | ||
− | <p id="pic_illustration"> | + | <p id="pic_illustration" style="text-align:center"> |
<strong>Figure 8.</strong> The simple EBFC 1.0 made by ourselves. | <strong>Figure 8.</strong> The simple EBFC 1.0 made by ourselves. | ||
</p> | </p> | ||
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<p> | <p> | ||
− | Due to the fact that there existed leakage phenomenon, after several tests, the voltage is basically 0V. After a resistance measure by the multimeter, we found that the battery’s internal resistance is too large, so we need to further improve the device. | + | Due to the fact that there existed leakage phenomenon, after several tests, the voltage is basically 0V. After a resistance measure by the multimeter, we found that the battery’s internal resistance is too large, so we need to further improve the device.<br><br> |
</p> | </p> | ||
<div class="project_pic"> | <div class="project_pic"> | ||
<p id="pic_title"><strong>2. The improved EBFC 2.0.</strong> We purchased the device from the internet. The middle of the device is proton exchange membrane, and the electrode material is carbon paper (Fig. 9A). We assembled the materials above and made the EBFC 2.0 (Fig. 9B).</p> | <p id="pic_title"><strong>2. The improved EBFC 2.0.</strong> We purchased the device from the internet. The middle of the device is proton exchange membrane, and the electrode material is carbon paper (Fig. 9A). We assembled the materials above and made the EBFC 2.0 (Fig. 9B).</p> | ||
<img src="https://static.igem.org/mediawiki/2015/c/cc/CHINA_CD_UESTC_RESULTfin09.png" width="60%"> | <img src="https://static.igem.org/mediawiki/2015/c/cc/CHINA_CD_UESTC_RESULTfin09.png" width="60%"> | ||
− | <p id="pic_illustration"> | + | <p id="pic_illustration" style="text-align:center"> |
<strong>Figure 9.</strong> The diagram of the EBFC 2.0. | <strong>Figure 9.</strong> The diagram of the EBFC 2.0. | ||
</p> | </p> | ||
− | </div> | + | </div><br><br> |
<p> | <p> | ||
− | <strong>EBFC performances.</strong> We added components into the device and test the performance with oscilloscope (Fig. 10). | + | <strong>EBFC performances.</strong> We added components into the device and test the performance with oscilloscope (Fig. 10).The figure 10A and B were control. |
</p> | </p> | ||
<div class="project_pic"> | <div class="project_pic"> | ||
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<p> | <p> | ||
From the Figure 10, we can see that the EBFC effected the desired result—voltage appearred. The voltage reached a stable level of about 160mV and lasted for about 40h. The highest voltage reached 0.25V. But the battery’s internal resistance was too large, resulting in small electric current, so we need to further improve the battery. | From the Figure 10, we can see that the EBFC effected the desired result—voltage appearred. The voltage reached a stable level of about 160mV and lasted for about 40h. The highest voltage reached 0.25V. But the battery’s internal resistance was too large, resulting in small electric current, so we need to further improve the battery. | ||
− | </p> | + | </p><br> |
<div class="project_pic" id="3Ddevice"> | <div class="project_pic" id="3Ddevice"> | ||
<p id="pic_title"><strong>3. The improved EFBC 3.0.</strong> In order to reduce the internal resistance and lower the cell cost, we designed the device 3.0 (Fig. 11). | <p id="pic_title"><strong>3. The improved EFBC 3.0.</strong> In order to reduce the internal resistance and lower the cell cost, we designed the device 3.0 (Fig. 11). | ||
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</div> | </div> | ||
<p>Run the 3D printing device and test, it didn’t achieve the desired result; the internal resistance of the battery didn’t get smaller. Apart from reducing the internal resistance, we have to improve the electrons transfer efficiency and make enzyme catalyze substrates constantly. So we need to find a better method to make enzyme gathered on the surface of two electrodes and immobilized.</p> | <p>Run the 3D printing device and test, it didn’t achieve the desired result; the internal resistance of the battery didn’t get smaller. Apart from reducing the internal resistance, we have to improve the electrons transfer efficiency and make enzyme catalyze substrates constantly. So we need to find a better method to make enzyme gathered on the surface of two electrodes and immobilized.</p> | ||
− | <p><strong>Next, we put | + | <p><strong>Next, we put MamW+RFP+laccase into our EBFC, the work is going on and detailed results may be presented on our PPT.</strong></p> |
</div> | </div> | ||
</div> | </div> | ||
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<h2>Magnetosome formation in <i>E.coli</i></h2> | <h2>Magnetosome formation in <i>E.coli</i></h2> | ||
<div class="project_pic"> | <div class="project_pic"> | ||
− | <p id="pic_title"><strong>1. Amplification of <i>mamAB.</i></strong> We separated <i>mamAB</i> into 3 parts and amplified each one by common PCR (Fig. 12A). We subcloned the three parts into | + | <p id="pic_title"><strong>1. Amplification of <i>mamAB.</i></strong> We separated <i>mamAB</i> into 3 parts and amplified each one by common PCR (Fig. 12A). We subcloned the three parts into pET-28a vector successfully, and named piGEM-AB. We verified it using digestion (Fig. 12B) and sequencing.</p> |
<img src="https://static.igem.org/mediawiki/2015/8/82/CHINA_CD_UESTC_RESULTfin12.png" width="60%"> | <img src="https://static.igem.org/mediawiki/2015/8/82/CHINA_CD_UESTC_RESULTfin12.png" width="60%"> | ||
<p id="pic_illustration"> | <p id="pic_illustration"> | ||
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<strong>Figure 15.</strong> Transmission electron microscopy images of modified <i>E.coli.</i> <strong>(A) (B)</strong> Images of cells of <i>BL21(DE3)</i> without any vectors prepared on a TEM grid. <strong>(C)</strong> Images of cells of <i>BL21(DE3)</i> transferred with piGEM-AB. <strong>(D)</strong> Images of cells of <i>BL21(DE3)</i> co-transferred with piGEM-AB and piGEM-G6. | <strong>Figure 15.</strong> Transmission electron microscopy images of modified <i>E.coli.</i> <strong>(A) (B)</strong> Images of cells of <i>BL21(DE3)</i> without any vectors prepared on a TEM grid. <strong>(C)</strong> Images of cells of <i>BL21(DE3)</i> transferred with piGEM-AB. <strong>(D)</strong> Images of cells of <i>BL21(DE3)</i> co-transferred with piGEM-AB and piGEM-G6. | ||
<strong>(E)-(H)</strong> Images of cells of <i>BL21(DE3)</i> co-transferred with piGEM-AB and piGEM-G6X. Arrows indicate the magnetosome. The scale bar corresponds to 200nm. | <strong>(E)-(H)</strong> Images of cells of <i>BL21(DE3)</i> co-transferred with piGEM-AB and piGEM-G6X. Arrows indicate the magnetosome. The scale bar corresponds to 200nm. | ||
− | </p> | + | </p><br> |
<p>From Fig. 14, we can see that we co-transformed piGEM-AB and piGE-G6X into <i>E.coli</i> successfully. After further culture in large scale, we observed cells with TEM. From Fig. 15 we can see that <i>E.coli</i>’s shape changing with the increase of operons. After transforming all of the four operons, we saw black particles like megnetosomes. But we learned that inclusion bodies are also black particles when observing under a TEM. So we ran SDS-PAGE, however we didn’t find any specific band, which indicated the black particles (Fig. 15F, G, H) could well be magnetosomes. We further observed that <i>E.coli</i> with these black particles were mostly in the decline stage. So we need to do more experiments, on the one hand, to confirm whether the black particles are magnetosomes, and on the other hand, to make <i>E.coli</i> produce magnetosomes stably. Then we can use these magnetosomes to realize laccases’ enrichment and immobilization. | <p>From Fig. 14, we can see that we co-transformed piGEM-AB and piGE-G6X into <i>E.coli</i> successfully. After further culture in large scale, we observed cells with TEM. From Fig. 15 we can see that <i>E.coli</i>’s shape changing with the increase of operons. After transforming all of the four operons, we saw black particles like megnetosomes. But we learned that inclusion bodies are also black particles when observing under a TEM. So we ran SDS-PAGE, however we didn’t find any specific band, which indicated the black particles (Fig. 15F, G, H) could well be magnetosomes. We further observed that <i>E.coli</i> with these black particles were mostly in the decline stage. So we need to do more experiments, on the one hand, to confirm whether the black particles are magnetosomes, and on the other hand, to make <i>E.coli</i> produce magnetosomes stably. Then we can use these magnetosomes to realize laccases’ enrichment and immobilization. | ||
</p> | </p> | ||
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<p>Time fleeting, five months passed by quickly, we all team members worked more than hardly. Fortunately, our efforts had been rewarded. We modified laccase and assembled our EBFC 1.0, EBFC 2.0 and EBFC 3.0 successfully. What’s more, on the way of immobilizing laccase using magnetosomes, we made great progress. We hope that our work could attract your eyesight. | <p>Time fleeting, five months passed by quickly, we all team members worked more than hardly. Fortunately, our efforts had been rewarded. We modified laccase and assembled our EBFC 1.0, EBFC 2.0 and EBFC 3.0 successfully. What’s more, on the way of immobilizing laccase using magnetosomes, we made great progress. We hope that our work could attract your eyesight. | ||
</p> | </p> | ||
− | <p | + | <p>We constructed the 16 vectors successfully through the five months (Table 1). |
</p> | </p> | ||
<p> | <p> | ||
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<td>piGEM-R-Lac</td> | <td>piGEM-R-Lac</td> | ||
<td><i>RFP</i>+<i>laccase</i></td> | <td><i>RFP</i>+<i>laccase</i></td> | ||
− | <td>Encode a fusion protein which makes | + | <td>Encode a fusion protein which makes laccase visible</td> |
<td><a href="http://parts.igem.org/Part:BBa_K1779204">BBa_K1779204</a></td> | <td><a href="http://parts.igem.org/Part:BBa_K1779204">BBa_K1779204</a></td> | ||
</tr> | </tr> | ||
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<td><i>mamW</i>+<i>RFP</i>+<i>laccase</i></td> | <td><i>mamW</i>+<i>RFP</i>+<i>laccase</i></td> | ||
<td> | <td> | ||
− | Encode a fusion protein to bind | + | Encode a fusion protein to bind laccase to the transmembrane protein MamW, and RFP can make it visible |
</td> | </td> | ||
<td><a href="http://parts.igem.org/Part:BBa_K1779200">BBa_K1779200</a><br> | <td><a href="http://parts.igem.org/Part:BBa_K1779200">BBa_K1779200</a><br> |
Latest revision as of 02:48, 19 September 2015
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RESULTS
Finally, we got the fusion proteins MamW + RFP + laccase and RFP + laccase. The fusion protein RFP + laccase worked very well in our EBFC. What's more, after we co-transferred the two vectors piGEM-AB and piGEM-G6X into Escherichia coli BL21(DE3), there were something interesting appeared in the bacteria!