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

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            <p>
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<B>RESULTS</B>
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                <B>RESULTS</B>
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&nbsp;&nbsp;We present details on the various methods such as vectors design, domain linker selection and choose of enzyme insertion site that used in the experiment on this page, if you are willing to check or follow our work, you can scan the methods here. Any questions or advice to us are acceptable at any time.
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                &nbsp;&nbsp;We present details on the various methods such as vectors design, domain linker selection and choose of enzyme insertion site that used in the experiment on this page, if you are willing to check or follow our work, you can scan the methods here. Any questions or advice to us are acceptable at any time.
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<h3>Part1.</h3>
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                            <h3>Laccase</h3>
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                            <div class="project_pic">
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                                <p id="pic_title">
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We separated mamAB into 3 parts and amplified each one by PCR.
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                                    1. We respectively amplified mamW, RFP and Laccase by common PCR and then we respectively combined W+R+L and R+L by fusion PCR.
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                                </p>
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<img src="https://static.igem.org/mediawiki/2015/4/48/CHINA_CD_UESTC_RESULT06.png" width="30%">
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Figure 1.1: We amplified three parts of mamAB by PCR using high fidelity polymerase Advantage.
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                                    Figure 1. The image of agarose gel electrophoresis. <strong>(A)</strong>
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</p>
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                                    M: DNA marker, mamW and RFP and laccase were amplified by PCR using high fidelity DNA polymerase. <strong>(B)</strong>
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                                    M: DNA marker, W+R+L: mamW+RFP+Laccase, R+L: RFP+ Laccase.
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                                </p>
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We used three parts to constructed mamAB successfully by subclone and we have verified them using digestion (Fig.1) and sequencing.
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                                    2. We purified the PCR products and successfully inserted the fragments into pACYCDuet-1, and named piGEM-WRL and piGEM-RL. We verified them using digestion and sequencing.
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                                </p>
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<img src="https://static.igem.org/mediawiki/2015/d/d6/CHINA_CD_UESTC_RESULT09.png" width="30%">
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                                <img src="https://static.igem.org/mediawiki/2015/d/d6/CHINA_CD_UESTC_RESULT09.png" width="30%">
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<p id="pic_illustration">
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                                <p id="pic_illustration">
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Figure 1.2: We confirmed mamAB by enzyme digestion using ApaI、SapI、NotI enzymes
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                                    Figure 2. Verification of vectors using digestion.
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                                    <strong>(A)</strong>
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                                    M: DNA marker. Lane 1, the piGEM-WRL without restriction endonuclease. Lane 2, the piGEM-WRL was digested by PstI +XhoI.
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                                    <strong>(B)</strong>
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                                    M: DNA marker. Lane 3, the piGEM-RL without restriction endonuclease. Lane 4, the piGEM-RL was digested by PstI +XhoI.
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We also constructed mamGFDC+mms6, and mamXY which are related to the formation of magnetosome.
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                                </p>
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                            </div>
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<img src="https://static.igem.org/mediawiki/2015/a/a3/CHINA_CD_UESTC_RESULT07.png" width="30%">
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                                <p id="pic_title">
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                                    3. We transformed the piGEM-RL into BL21(DE3) and conducted inducible expression. The concentration and color of bacterium liquid are as followed:
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                                </p>
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                                <img src="https://static.igem.org/mediawiki/2015/a/a3/CHINA_CD_UESTC_RESULT07.png" width="30%">
  
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Figure 1.3: We amplified mamGFDC+mms6 and mamXY by PCR using high fidelity polymerase Advantage.
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                                    Figure 3. The color and the concentration of bacterium liquid.
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                                    <strong>(A)</strong>
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                                    The color degree among different bacterium liquid. The higher concentration of bacterium liquid showed redder.
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                                    <strong>(B)</strong>
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<p id="pic_title">This is part has relation with the formation of magnetosome</p>
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                                    The OD <sub>600</sub>
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<img src="https://static.igem.org/mediawiki/2015/c/c0/CHINA_CD_UESTC_RESULT10.png" width="30%">
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                                    of different concentration of baterium liquid at different time using ultraviolet spectrophotometer.
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                                </p>
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                            </div>
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                                <p id="pic_title">4. The SDS-PAGE of the cell with the piGEM-RL.</p>
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                                <img src="https://static.igem.org/mediawiki/2015/c/c0/CHINA_CD_UESTC_RESULT10.png" width="30%">
  
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<p id="pic_illustration">
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                                <p id="pic_illustration">
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Figure 1.4: We confirmed piGEM-G6X by enzyme digestion using Hind III enzymes in Lane1 and Apal I and Pst I enzymes in Lane2.
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                                    Figure 4. Testing expression of RFP+Laccase in E.coli. M: marker. Lane 1, bacteria untransformed. Lane 2, Bacteria which contain piGEM-RL. Induced at 37centigrades, 180rpm, 10 hours with 0.5mM IPTG.Figure 1.4: We confirmed piGEM-G6X by enzyme digestion using Hind III enzymes in Lane1 and Apal I and Pst I enzymes in Lane2.
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                            <p>
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<p id="pic_title">We respectively amplified three fragments by PCR.</p>
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                                In the gel we found that the band located near 80KDa is RFP+Laccase.
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                            </p>
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                                <p id="pic_title">
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                                    5. The activity of laccase：
 +
                                    <br>
 +
                                    Using Ultrasonic Cell Disruptor to crush the bacterium in ice-bath. Collect the supernatant and detect the activity of laccase by ABTS method.
 +
                                </p>
  
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<img src="https://static.igem.org/mediawiki/2015/d/d5/CHINA_CD_UESTC_RESULT08.png" width="30%">
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                                <img src="https://static.igem.org/mediawiki/2015/d/d5/CHINA_CD_UESTC_RESULT08.png" width="30%">
  
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<p id="pic_illustration">
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                                <p id="pic_illustration">
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Figure 1.5: We amplified mamW and RFP and Laccase by PCR using high fidelity polymerase Advantage.
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                                    Figure 5. The color of supernate and activity of laccase.
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</p>
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                                    <strong>(A)</strong>
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                                    The higher concentration of laccase showed redder.
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<div class="project_pic">
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                                    <strong>(B)</strong>
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<p id="pic_title">
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                                    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 time.
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We successfully connected three fragments together by fusion PCR
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                                </p>
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                            </div>
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<img src="https://static.igem.org/mediawiki/2015/0/05/CHINA_CD_UESTC_RESULT11.png" width="30%">
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<p id="pic_illustration">
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Figure 1.6: We amplified mamW+RFP+Laccase and RFP+ Laccase by fusion PCR using high fidelity polymerase Advantage.
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                                    6. We transformed piGEM-WRL into BL21(DE3) and conducted inducible expression. We got a series of samples which was cultivated at different time and chose one to compare with BL21(DE3) untransformed.
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                                <img src="https://static.igem.org/mediawiki/2015/0/05/CHINA_CD_UESTC_RESULT11.png" width="30%">
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                                    Figure 6. The color of supernate and activity of laccase.
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We recovered the PCR products and succeddfully inserted the fragment into pACYCDuet. and confirmed piGEM-WRL and piGEM-RL by restriction digestion.
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                                    <strong>(A)</strong>
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</p>
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                                    The left is BL21(DE3) untransformed and the right is BL21(DE3) transformed with piGEM-WRL.
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                                    <strong>(B)</strong>
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                                    The activity of MamW+RFP+Laccase.
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                                </p>
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                            </div>
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                            <div class="project_pic">
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                                <p id="pic_title">In the end, we compared the two enzyme activity curves.</p>
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                                <img src="https://static.igem.org/mediawiki/2015/0/05/CHINA_CD_UESTC_RESULT11.png" width="30%">
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                                <p id="pic_illustration">
 +
                                    Figure 7. The comparation of two laccases activity. The red curve represented piGEM-RL and the blue curve presented piGEM-WRL.
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                                </p>
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                            </div>
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                            <p>
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                                This picture showed that the enzyme activity of the laccase coded by piGEM-RL is higher than another. Next we put the RFP+Laccase in the EBFC.
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Figure 1.7: We confirmed piGEM-WRL by enzyme digestion using Pst1 +Xho1 enzymes
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Figure 1.8: We confirmed piGEM-RL by enzyme digestion using Pst1 +Xho1 enzymes
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                            <h3>Enzymatic biofuel cell (EBFC)</h3>
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                            <div class="project_pic">
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                                <p id="pic_title">1. Device 1：EBFC 1.0</p>
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                                <img src="https://static.igem.org/mediawiki/2015/2/2f/CHINA_CD_UESTC_RESULT01.jpg" width="40%" style="margin-left:100px;">
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 +
                                    Figure 8. The simple EBFC 1.0 made by ourselves.<strong>(A)</strong> Make this device using discarded bottles with the proton exchange membrane in the middle.<strong>(B)</strong>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.
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                                Due to the fact that the sealing was not very good and 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.
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                                <p id="pic_title">2. Device 2: EBFC 2.0</p>
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                                <img src="https://static.igem.org/mediawiki/2015/f/f2/CHINA_CD_UESTC_RESULT14.jpg" width="40%" style="margin-left:100px;">
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                                <p id="pic_illustration">
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                                    Figure 9. The diagram of the EBFC 2.0. Purchase the device from the internet.<strong>(A)</strong>The middle of the device is proton exchange membrane; the electrode material is carbon paper.<strong>(B)</strong>The diagram of the fixed EBFC 2.0.
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                                </p>
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                                <p id="pic_title"></p>
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                                <img src="https://static.igem.org/mediawiki/2015/7/74/CHINA_CD_UESTC_RESULT15.png" width="40%" style="margin-left:100px;">
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                                <p id="pic_illustration">
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                                    Figure 10. The EBFC 2.0 performances.<strong>(A)</strong>Before adding enzyme, the voltage was 0V.<strong>(B)</strong>the scan map of oscilloscope of the left.<strong>(C)</strong>After adding enzyme, the voltage increased instantly but the voltage was far from stable. After about 5 minutes, the voltage got stable.<strong>(D)</strong>The voltage reached a stable level of about 160mV and lasted for about 40h. The scan time was 50s and every grid represents 100mV.
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                                </p>
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                            </div>
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                            <p>
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                                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 the small electric current, so we need to further improve the battery.
 +
                            </p>
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                                <p id="pic_title">3. Device 3 EFBC 3.0</p>
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                                <img src="https://static.igem.org/mediawiki/2015/5/5d/CHINA_CD_UESTC_RESULT16.png" width="40%" style="margin-left:100px;">
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                                <p id="pic_illustration">
 +
                                    Figure 11. The device made by 3D print. Add the components through the upper holes and fix the electrodes to the edge of the holes.
 +
                                </p>
 +
                            </div>
 +
                            <p>Run the 3D print device and test, it didn’t achieve the desired result; the internal resistance of the battery didn’t get smaller. Apart from lowering 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 gather to the surface of two electrodes and immobilized.</p>
 +
                        </div>
 +
                    </div>
 +
                </div>
 +
            </div>
  
−
<h3>Part 2.</h3>
+
            <div class="slide" id="slide2" data-slide="2" data-stellar-background-ratio="0.5" style="background-position: 0px 669px;">
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<p>
+
                <div class="container clearfix">
−
After our experiments, we successfully combine mamW,Laccase and RFP. We measured different OD of bacterium at different time, and record the color. We also detected the activity of Laccase. We found that the activity of Laccase has positive correlation with OD and the color. We make sure that the activity of Laccase have the catalytic property by using ABTS method. So that we can put them at cathode of EBFC and make it work.
+
                    <div id="content">
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</p>
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                        <div class="grid_8">
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<div class="clear"></div>
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<p> <strong>(1)SDS-PAGE</strong>
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</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/2015/2/2f/CHINA_CD_UESTC_RESULT01.jpg" width="40%" style="margin-left:100px;">
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<p id="pic_illustration">
+
−
Lane1 :control of pACYCDuet-1-RFP-Laccasse
+
−
<br>
+
−
Lane2 :BL21 Bacterial strain which contain pACYCDuet-1-RFP-Laccasse induced expression.
+
−
</p>
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</div>
+
  
−
<p>
+
                            <h3>Magnetosome</h3>
−
Induction fonditions:37centigrades，200rpm，induce 10 hours with IPTG  which final concentration is 0.5mM. Using Ultrasonic Cell Disruptor to crush the bacterium in ice-bath.
+
                            <div class="project_pic">
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</p>
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                                <p id="pic_title">1. We separated <i>mamAB</i> into 3 parts and amplified each one by common PCR. We subcloned the three parts into pET28a vector successfully, and named piGEM-AB. We verified it using digestion and sequencing.</p>
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<p>
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                                <img src="https://static.igem.org/mediawiki/2015/a/ad/CHINA_CD_UESTC_RESULT19.png" width="40%" style="margin-left:100px;">
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Add 6*SDS-PAGE loading buffer，put it in boiling water bath for 5minius . The size of the target protein is 84KDa which is fusion expressed by RFP+Laccse,In gel we found that the line Bacterial strain which contain pACYCDuet-1-RFP-Laccasse is located near 80KDa, just as we expected.
+
                                <p id="pic_illustration">
−
</p>
+
                                    Figure 12. The construction of piGEM-AB. <strong>(A)</strong> The three parts of mamAB by PCR using high fidelity DNA polymerase. M: marker. Lane 1, <i>mamAB</i> part1. Lane 2, <i>mamAB</i> part2. Lane 3, <i>mamAB</i> part3.<strong>(B)</strong>M: marker. Lane 4, digestion the plasmid piGEM-AB by restriction endonuclease ApaI,SapI,NotI.
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<div class="clear"></div>
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                                </p>
−
<p> <strong>(2)Bacterium liquid</strong>
+
                            </div>
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</p>
+
                            <div class="project_pic">
−
<p>concentration of bacterium detected at different time</p>
+
                                <p id="pic_title">2. We also amplified <i>mamGFDC+mms6</i>, and <i>mamXY</i> by common PCR. We cloned the two parts into pCDFDuet-1 vector successfully, and named piGEM-G6X. We verified it using digestion and sequencing.</p>
 +
                                <img src="https://static.igem.org/mediawiki/2015/2/2a/CHINA_CD_UESTC_RESULT20.png" width="60%" style="margin-left:100px;">
 +
                                <p id="pic_illustration">
 +
                                    Figure 13. The construction of piGEM-G6X.<strong>(A)</strong> amplify <i>mamGFDC+mms6</i> and <i>mamXY</i> by PCR using high fidelity DNA polymerase. M: marker. Lane 1, <i>mamGFDC+mms6</i>. Lane 2, <i>mamXY</i>. (B) M: marker. Lane 3, digestion the plasmid piGEM-G6X by restriction endonuclease HindIII. Lane 4, digestion the plasmid piGEM-G6X by restriction endonuclease ApalI, PstI.
 +
                                </p>
 +
                            </div>
  
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<div class="project_two_line">
+
                            <div class="project_pic">
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<div class="project_left_pic">
+
                                <p id="pic_title">3. We constructed the piGEM-AB and piGEM-G6X successfully and co-transferred them into <i>E.coli</i>, and detected by the colony PCR.</p>
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<img src="https://static.igem.org/mediawiki/2015/6/69/CHINA_CD_UESTC_RESULT02.jpg" width="340px" height="240px">
+
                                <img src="https://static.igem.org/mediawiki/2015/6/6b/CHINA_CD_UESTC_RESULT23.png" width="40%" style="margin-left:100px;">
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<p id="pic_illustration">
+
                                <p id="pic_illustration">Figure 14. The bacterial colony PCR result. M: marker. Lane 1, the colony with piGEM-AB. Lane 2 and 3, the colony with piGEM-AB+piGEM-G6. Lane 4 and 5, the colony with piGEM-AB+piGEM-G6X.</p>
−
Figure 2.2: We compared color degree among different bacterium  liquid. The higher concentration of bacterium showed redder.
+
                            </div>
−
</p>
+
                            <div class="project_pic">
−
</div>
+
                                <p id="pic_title">4. Transmission electron microscope (TEM)</p>
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<div class="project_right_pic">
+
                                <img src="https://static.igem.org/mediawiki/2015/6/6b/CHINA_CD_UESTC_RESULT23.png" width="40%" style="margin-left:100px;">
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<img src="https://static.igem.org/mediawiki/2015/3/3d/CHINA_CD_UESTC_RESULT03.png" width="340px" height="240px">
+
                                <p id="pic_illustration">Figure 15. Transmission electron microscopy images of modified E.coli. <strong>(A) (B)</strong>Images of cells of BL21(DE3) without any vectors prepared on a TEM grid.<strong>(C)</strong> Images of cells of BL21(DE3) transferred with piGEM-AB.<strong>(D)</strong> Images of cells of BL21(DE3) co-transferred with piGEM-AB and piGEM-G6.
−
<p id="pic_illustration">
+
                                <strong>(E)-(H)</strong> Images of cells of BL21(DE3) co-transferred with piGEM-AB and piGEM-G6X. Arrows indicate the magnetosome. The scale bar corresponds to 200 nm.
−
Figure 2.3: We measured the OD of different concentration of baterium at different time  using ultraviolet spectrophotometer
+
                                            </p>
−
</p>
+
                            </div>
−
</div>
+
                        </div>
−
</div>
+
                    </div>
 +
                </div>
 +
            </div>
  
−
<div class="clear"></div>
+
            <div class="slide" id="slide2" data-slide="2" data-stellar-background-ratio="0.5" style="background-position: 0px 669px;">
−
<p>
+
                <div class="container clearfix">
−
<strong>(3)Supernatant after crush：</strong>
+
                    <div id="content">
−
</p>
+
                        <div class="grid_8">
−
<p>
+
−
Using Ultrasonic Cell Disruptor to crush the bacterium in ice-bath. Collect the Supernatant and detect the activity of Laccase  by using ABTS. The conclusion is that as the time pass the Laccasse is more.
+
−
</p>
+
  
−
<div class="project_two_line">
+
                            <h3>Summary</h3>
−
<div class="project_left_pic">
+
                            <p>
−
<img src="https://static.igem.org/mediawiki/2015/8/84/CHINA_CD_UESTC_RESULT04.jpg" width="340px" height="240px">
+
                                <strong>Table 1. the 16 vectors that we constructed successfully through the three months.</strong>
−
<p id="pic_illustration">
+
                            </p>
−
Figure 2.4: We broke the bacterium at different time and extracted the supernate and compared color degree among different bacterium  liquid. The higher concentration of bacterium showed redder.
+
−
</p>
+
−
</div>
+
−
<div class="project_right_pic">
+
−
<img src="https://static.igem.org/mediawiki/2015/5/56/CHINA_CD_UESTC_RESULT05.png" width="340px" height="240px">
+
−
<p id="pic_illustration">
+
−
Figure 2.5: We determined enzymes activities from different supernate using ABTS method. Every supernate presents the different bacterium which are cultivated for different time.
+
−
</p>
+
−
</div>
+
−
</div>
+
  
−
<p>
+
                            <table class="table1">
−
<strong>mamW+RFP+Laccase</strong>
+
                                <thead>
−
</p>
+
                                    <tr>
−
<p>
+
                                        <th></th>
−
We transferred piGEM-WRL into BL21 bacterial strain and got a series of bacter-ium liquid which are cultivated for different time.
+
                                        <th scope="col" abbr="Starter" class="table-showfull">Vectors</th>
−
</p>
+
                                        <th scope="col" abbr="Medium">Inserted gene</th>
−
<p>
+
                                        <th scope="col" abbr="Business">Gene function</th>
−
<strong>Bacterium liquid</strong>
+
                                        <th scope="col">Related parts</th>
−
</p>
+
                                    </tr>
−
<p>
+
                                </thead>
−
We compared one of the experimental groups with control group and the experimental group showed red color which indicated the this expressed successfully. But the color is dim compared with the enzyme coded by piGEM-RL which indicated the enzyme coded by piGEM-WRL activity is weaker.
+
                                <tbody>
−
</p>
+
                                    <tr>
−
<div class="project_pic">
+
                                        <th scope="row">1</th>
−
<p id="pic_title"></p>
+
                                        <td>piGEM-AB</td>
−
<img src="https://static.igem.org/mediawiki/2015/f/f2/CHINA_CD_UESTC_RESULT14.jpg" width="40%" style="margin-left:100px;">
+
                                        <td>mamAB</td>
−
<p id="pic_illustration">
+
                                        <td>
−
Figure 2.6: We choose one to compared with BL21 bacterial strain containing empty vector as control group and showed that experimental group is redder than control group.
+
                                            Encode a series of protein that is essential for magnetosome synthesis
−
</p>
+
                                        </td>
−
</div>
+
                                        <td><a>BBa_K1779205</a><br>
−
<p>
+
                                        <a>BBa_K1779206</a><br>
−
We broke the bacterium liquid and extracted the supernate. Then we measured the activities of enzyme from different supernate with the same method.
+
                                        <a>BBa_K1779207</a><br>
−
</p>
+
                                        <a>BBa_K1779208</a><br>
−
<div class="project_pic">
+
                                        <a>BBa_K1779209</a><br>
−
<p id="pic_title"></p>
+
                                        <a>BBa_K1779210</a><br>
−
<img src="https://static.igem.org/mediawiki/2015/7/74/CHINA_CD_UESTC_RESULT15.png" width="40%" style="margin-left:100px;">
+
                                        <a>BBa_K1779211</a><br>
−
<p id="pic_illustration">
+
                                        <a>BBa_K1779212</a><br>
−
Figure 2.7: We determined enzymes activities from different supernate using ABTS method. Every supernate presents the different bacterium which are cultivated for different time.
+
                                        <a>BBa_K1779213</a><br>
−
</p>
+
                                        <a>BBa_K1779214</a><br>
−
</div>
+
                                        </td>
−
<p>
+
                                    </tr>
−
In the end, we compared the two enzyme activity curves. The one represented the enzyme coded by piGEM-RL, the other is coded by piGEM-WRL. This picture showed that the enzyme activity of the laccase coded by piGEM-RL is higher than the other.
+
                                    <tr>
−
</p>
+
                                        <th scope="row">2</th>
−
<div class="project_pic">
+
                                        <td>piGEM-G6</td>
−
<p id="pic_title"></p>
+
                                        <td>mamGFDC+mms6</td>
−
<img src="https://static.igem.org/mediawiki/2015/5/5d/CHINA_CD_UESTC_RESULT16.png" width="40%" style="margin-left:100px;">
+
                                        <td>
−
<p id="pic_illustration">
+
                                            Encode a series of proteins that can regulate the size and shape of crystals in the formation of magnetosome
−
Figure 2.8: two enzyme activity curves presented the two different laccases coded by different vector. the red curve represented piGEM-RL and the blue curve presented piGEM-WRL.
+
                                        </td>
−
</p>
+
                                        <td><a>BBa_K1779100</a></td>
−
</div>
+
                                    </tr>
−
</div>
+
                                    <tr>
−
</div>
+
                                        <th scope="row">3</th>
−
</div>
+
                                        <td>piGEM-G6X</td>
−
</div>
+
                                        <td>mamGFDC+mms6+mamXY</td>
 +
                                        <td>
 +
                                            Encode a series of proteins that can regulate the size and shape of crystals in the formation of magnetosome
 +
                                        </td>
 +
                                        <td><a>BBa_K1779100</a><br>
 +
                                        <a>BBa_K1779101</a>
 +
</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <th scope="row">4</th>
 +
                                        <td>piGEM-R-Lac</td>
 +
                                        <td>RFP+laccase</td>
 +
                                        <td>Encode a fusion protein which makes laccase visible</td>
 +
                                        <td><a>BBa_K1779204</a></td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <th scope="row">5</th>
 +
                                        <td>piGEM-W-R-Lac</td>
 +
                                        <td>mamW+RFP+laccase</td>
 +
                                        <td>
 +
                                            Encode a fusion protein to bind laccase to the transmembrane protein MamW, and RFP can make it visible
 +
                                        </td>
 +
                                        <td><a>BBa_K1779200</a><br>
 +
                                            <a>BBa_K1779201</a><br>
 +
                                            <a>BBa_K1779202</a><br>
 +
                                            <a>BBa_K1779203</a><br>
 +
</td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <th scope="row">6</th>
 +
                                        <td>piGEM-Plac-H</td>
 +
                                        <td>LacI promoter+mamH</td>
 +
                                        <td>
 +
                                            To make sure whether mamH gene can successfully express
 +
                                        </td>
 +
                                        <td></td>
 +
                                    </tr>
 +
                                    <tr>
 +
                                        <th scope="row">7</th>
 +
                                        <td>piGEM-Plac-G</td>
 +
                                        <td>lac promoter+mamG</td>
 +
                                        <td>To make sure whether mamG gene can successfully express</td>
 +
                                        <td></td>
  
−
<div class="slide" id="slide2" data-slide="2" data-stellar-background-ratio="0.5" style="background-position: 0px 669px;">
+
                                    </tr>
−
<div class="container clearfix">
+
                                    <tr>
−
<div id="content">
+
                                        <th scope="row">8</th>
−
<div class="grid_8">
+
                                        <td>piGEM-Plac-6</td>
 +
                                        <td>lac promoter+mms6</td>
 +
                                        <td>To make sure whether mamH gene can successfully express</td>
 +
                                        <td></td>
  
−
<h3>Part 3.</h3>
+
                                    </tr>
−
<p>
+
                                    <tr>
−
In order to improve the catalytic efficiency of laccase, we decided to immobilize this enzyme. After looking over lots of methods to immobilize laccase. We finally decide to bind laccase to magnetosome. To bind laccase to magnetosome, we designed a series of experiments to let Escherichia coli to express the proteins which are related with the information of the magnetosome, and to get magnetosomes. Through three mouth, we succeed to construct fifteen recombinant plasmids. Details are as follows:
+
                                        <th scope="row">9</th>
−
</p>
+
                                        <td>piGEM-Plac-Y</td>
−
<p>
+
                                        <td>lac promoter+mamY</td>
−
<strong>1.For expression</strong>
+
                                        <td>To make sure whether mamH gene can successfully express</td>
−
</p>
+
                                        <td></td>
  
−
<table class="table1">
+
                                    </tr>
−
<thead>
+
                                    <tr>
−
<tr>
+
                                        <th scope="row">10</th>
−
<th></th>
+
                                        <td>piGEM-PH-R-1</td>
−
<th scope="col" abbr="Starter" class="table-showfull">Vectors name</th>
+
                                        <td>mamH promoter+RFP</td>
−
<th scope="col" abbr="Medium">Inserted gene</th>
+
                                        <td>To make sure whether mamH promoter can work</td>
−
<th scope="col" abbr="Business">Gene function</th>
+
                                        <td></td>
−
</tr>
+
−
</thead>
+
−
<tbody>
+
−
<tr>
+
−
<th scope="row">1</th>
+
−
<td>piGEM-AB</td>
+
−
<td>mamAB</td>
+
−
<td>
+
−
Encode a series of protein that are essential for magnetosome synthesis
+
−
</td>
+
−
</tr>
+
−
<tr>
+
−
<th scope="row">2</th>
+
−
<td>piGEM-G6</td>
+
−
<td>mamGFDC+mms6</td>
+
−
<td>
+
−
Encode a series of protein that can regulate the size and shape of crystals in the formation of magnetosome
+
−
</td>
+
−
</tr>
+
−
<tr>
+
−
<th scope="row">3</th>
+
−
<td>piGEM-G6X</td>
+
−
<td>mamGFDC+mms6+mamXY</td>
+
−
<td>
+
−
Encode a series of protein that can regulate the size and shape of crystals in the formation of magnetosome
+
−
</td>
+
−
</tr>
+
−
<tr>
+
−
<th scope="row">4</th>
+
−
<td>piGEM-R-Lac</td>
+
−
<td>RFP+laccase</td>
+
−
<td>Encode a fusion protein which makes laccase visible</td>
+
−
</tr>
+
−
<tr>
+
−
<th scope="row">5</th>
+
−
<td>piGEM-W-R-Lac</td>
+
−
<td>mamW+RFP+laccase</td>
+
−
<td>
+
−
Encode a fusion protein to bind laccase to the transmembrane protein MamW, and RFP can make it visible
+
−
</td>
+
−
</tr>
+
−
<tr>
+
−
<th scope="row">6</th>
+
−
<td>piGEM-W-Lac</td>
+
−
<td>mamW+laccase</td>
+
−
<td>
+
−
Encode a fusion protein to bind laccase to the transmembrane protein MamW
+
−
</td>
+
−
</tr>
+
−
</tbody>
+
−
</table>
+
−
<p>
+
−
<strong>2.For detection</strong>
+
−
</p>
+
−
<table class="table1">
+
−
<thead>
+
−
<tr>
+
−
<th></th>
+
−
<th scope="col" abbr="Starter" class="table-showfull">Vectors name</th>
+
−
<th scope="col" abbr="Medium">Inserted gene</th>
+
−
<th scope="col" abbr="Business">Gene function</th>
+
−
</tr>
+
−
</thead>
+
−
<tbody>
+
−
<tr>
+
−
<th scope="row">1</th>
+
−
<td>piGEM-Plac-H</td>
+
−
<td>lac promoter+mamH</td>
+
−
<td>To make sure whether mamH gene can successfully express</td>
+
−
</tr>
+
−
<tr>
+
−
<th scope="row">2</th>
+
−
<td>piGEM-Plac-G</td>
+
−
<td>lac promoter+mamG</td>
+
−
<td>To make sure whether mamG gene can successfully express</td>
+
−
</tr>
+
−
<tr>
+
−
<th scope="row">3</th>
+
−
<td>piGEM-Plac-6</td>
+
−
<td>lac promoter+mms6</td>
+
−
<td>To make sure whether mamH gene can successfully express</td>
+
−
</tr>
+
−
<tr>
+
−
<th scope="row">4</th>
+
−
<td>piGEM-Plac-Y</td>
+
−
<td>lac promoter+mamY</td>
+
−
<td>To make sure whether mamH gene can successfully express</td>
+
−
</tr>
+
−
<tr>
+
−
<th scope="row">5</th>
+
−
<td>piGEM-PH-R-1</td>
+
−
<td>mamH promoter+RFP</td>
+
−
<td>To make sure whether mamH promoter can work</td>
+
−
</tr>
+
−
<tr>
+
−
<th scope="row">6</th>
+
−
<td>piGEM-PG-R</td>
+
−
<td>mamG promoter+RFP</td>
+
−
<td>To make sure whether mamG promoter can work</td>
+
−
</tr>
+
−
<tr>
+
−
<th scope="row">7</th>
+
−
<td>piGEM-P6-R</td>
+
−
<td>mms6 promoter+RFP</td>
+
−
<td>To make sure whether mms6 promoter can work</td>
+
−
</tr>
+
−
<tr>
+
−
<th scope="row">8</th>
+
−
<td>piGEM-PY-R</td>
+
−
<td>mamY promoter+RFP</td>
+
−
<td>To make sure whether mamY promoter can work</td>
+
−
</tr>
+
−
<tr>
+
−
<th scope="row">9</th>
+
−
<td>piGEM-PH-R-2</td>
+
−
<td>mamH promoter+RFP</td>
+
−
<td>To verify the backbone pET28a</td>
+
−
</tr>
+
−
<tr>
+
−
<th scope="row">10</th>
+
−
<td>piGEM-GFP-PG6-R</td>
+
−
<td>GFP+mamG-mms6 promoter+RFP</td>
+
−
<td>To make sure whether mamG-mms6 promoter can work</td>
+
−
</tr>
+
−
</tbody>
+
−
</table>
+
  
−
</div>
+
                                    </tr>
−
</div>
+
                                    <tr>
−
</div>
+
                                        <th scope="row">11</th>
−
</div>
+
                                        <td>piGEM-PG-R</td>
 +
                                        <td>mamG promoter+RFP</td>
 +
                                        <td>To make sure whether mamG promoter can work</td>
 +
                                        <td></td>
  
−
<div class="slide" id="slide2" data-slide="2" data-stellar-background-ratio="0.5" style="background-position: 0px 669px;">
+
                                    </tr>
−
<div class="container clearfix">
+
                                    <tr>
−
<div id="content">
+
                                        <th scope="row">12</th>
−
<div class="grid_8">
+
                                        <td>piGEM-P6-R</td>
 +
                                        <td>mms6 promoter+RFP</td>
 +
                                        <td>To make sure whether mms6 promoter can work</td>
 +
                                        <td></td>
  
−
<h3>Part 4.</h3>
+
                                    </tr>
−
<p>
+
                                    <tr>
−
<strong>Device 1：EBFC 1.0</strong>
+
                                        <th scope="row">13</th>
−
</p>
+
                                        <td>piGEM-PY-R</td>
−
<div class="project_two_line">
+
                                        <td>mamY promoter+RFP</td>
−
<div class="project_left_pic">
+
                                        <td>To make sure whether mamY promoter can work</td>
−
<img src="https://static.igem.org/mediawiki/2015/1/1a/CHINA_CD_UESTC_RESULT17.png" width="340px" height="240px">
+
                                        <td></td>
−
<p id="pic_illustration">
+
−
Figure 4.1. Make this device using discarded bottles with the proton exchange membrane in the middle.
+
  
−
</p>
+
                                    </tr>
−
</div>
+
                                    <tr>
−
<div class="project_right_pic">
+
                                        <th scope="row">14</th>
−
<img src="https://static.igem.org/mediawiki/2015/f/fd/CHINA_CD_UESTC_RESULT18.png" width="340px" height="240px">
+
                                        <td>piGEM-PH-R-2</td>
−
<p id="pic_illustration">
+
                                        <td>mamH promoter+RFP</td>
−
Figure 4.2. Fix the device 1 in the foam board, add each component (10 ml in total) according to the sketch. Use multimeter and oscilloscope to test voltage.
+
                                        <td>To make sure whether mamH promoter can work and verify the backbone of pET28a</td>
−
</p>
+
                                        <td></td>
−
</div>
+
−
</div>
+
−
<p>
+
−
Due to the fact that the sealing was not very good and there existed leakage phenomenon, after several tests, the voltage is basically 0. After a resistance measure by the multimeter, we found that the battery’s internal resistance is too high, so we need to further improve the device.
+
−
</p>
+
  
−
<p>
+
                                    </tr>
−
<strong>Device 2: EBFC 2.0</strong>
+
                                    <tr>
−
</p>
+
                                        <th scope="row">15</th>
−
<div class="project_pic">
+
                                        <td>piGEM-LacI-RFP</td>
−
<p id="pic_title"></p>
+
                                        <td>LacI promoter+RFP</td>
−
<img src="https://static.igem.org/mediawiki/2015/a/ad/CHINA_CD_UESTC_RESULT19.png" width="40%" style="margin-left:100px;">
+
                                        <td>To verify the backbone of pET28a</td>
−
<p id="pic_illustration">
+
                                        <td></td>
−
Figure 4.3. The middle of the device is proton exchange membrane; the electrode material is carbon paper. Run the device.
+
−
</p>
+
−
</div>
+
−
<div class="project_pic">
+
−
<p id="pic_title"></p>
+
−
<img src="https://static.igem.org/mediawiki/2015/2/2a/CHINA_CD_UESTC_RESULT20.png" width="60%" style="margin-left:100px;">
+
−
<p id="pic_illustration">
+
−
Figure 4.4. Before adding enzyme, the voltage is 0; the right is the scan map of oscilloscope in the left.
+
−
</p>
+
−
</div>
+
−
<p>After adding enzyme:</p>
+
−
<div class="project_two_line">
+
−
<div class="project_left_pic">
+
−
<img src="https://static.igem.org/mediawiki/2015/7/7f/CHINA_CD_UESTC_RESULT21.png" width="340px" height="240px">
+
−
<p id="pic_illustration">
+
−
Figure 4.5. the voltage increased instantly the moment when adding enzyme, but the voltage was far from stable. After about 5 minutes, the voltage gets stable.
+
−
</p>
+
−
</div>
+
−
<div class="project_right_pic">
+
−
<img src="https://static.igem.org/mediawiki/2015/4/49/CHINA_CD_UESTC_RESULT22.png" width="340px" height="240px">
+
−
<p id="pic_illustration">
+
−
Figure 4.7. the voltage reached a stable level of 155mV. The scan time is 50s and every grid represents 100mV.
+
−
</p>
+
−
</div>
+
−
</div>
+
−
<p>
+
−
From the upper picture, we can see that the EBFC effected the desired result—produced voltage, the highest voltage reached 0.25V and lasted for____. But the battery’s internal resistance was too large, resulting in the small electric current, so we need to further improve the battery.
+
−
</p>
+
  
−
<p>
+
                                    </tr>
−
<strong>Device 3: EFBC 3.0</strong>
+
                                        <tr>
−
</p>
+
                                        <th scope="row">16</th>
−
<div class="project_pic">
+
                                        <td>piGEM-GFP-PG6-R</td>
−
<p id="pic_title"></p>
+
                                        <td>GFP+mamG-mms6 promoter+RFP</td>
−
<img src="https://static.igem.org/mediawiki/2015/6/6b/CHINA_CD_UESTC_RESULT23.png" width="40%" style="margin-left:100px;">
+
                                        <td>To make sure whether mamG-mms6 promoter can work</td>
−
<p id="pic_illustration">Figure 4.8. 3D - print EBFC device.</p>
+
                                        <td></td>
−
<p>
+
−
Run the 3D print device and test, it didn’t achieve the desired result; the internal resistance of the battery didn't get smaller. In order to further improve the energy-producing effect and make enzyme catalyze substrates constantly, we need to find a better method to make enzyme gathered on the surface of two electrodes and fixed, so enzyme wouldn’t decrease with the change of substrates, realizing enzyme’s constant catalysis and improving the longevity of the battery.
+
−
</p>
+
  
−
<p>
+
                                    </tr>
−
<strong>Results of the promoter validation work:</strong>
+
                                </tbody>
−
</p>
+
                            </table>
−
<p>
+
−
In order to find the reason why magnetosomes canot form, we designed experiments to validate the promoters of 4 operons (connected to the design page).We successfully constructed the promoter validation work related vectors and transferred them to E. coli, cultured at 37 ℃and 180 RPM for 24 hours, but the bacteria liquid didn’t turn red. Then we broke cells and operate SDS-page electrophoresis, but we also didn’t find an RFP corresponding electrophoretic band. It meant that the native promoters related to the formation of magnetosomes in magnetotactic bacteria can’t work in E.coli, so finally we didn’t get magnetosomes.
+
−
</p>
+
−
</div>
+
  
−
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+
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−
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+
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Revision as of 16:48, 15 September 2015

<!DOCTYPE html>

RESULTS

  We present details on the various methods such as vectors design, domain linker selection and choose of enzyme insertion site that used in the experiment on this page, if you are willing to check or follow our work, you can scan the methods here. Any questions or advice to us are acceptable at any time.

Laccase

1. We respectively amplified mamW, RFP and Laccase by common PCR and then we respectively combined W+R+L and R+L by fusion PCR.

Figure 1. The image of agarose gel electrophoresis. (A) M: DNA marker, mamW and RFP and laccase were amplified by PCR using high fidelity DNA polymerase. (B) M: DNA marker, W+R+L: mamW+RFP+Laccase, R+L: RFP+ Laccase.

2. We purified the PCR products and successfully inserted the fragments into pACYCDuet-1, and named piGEM-WRL and piGEM-RL. We verified them using digestion and sequencing.

Figure 2. Verification of vectors using digestion. (A) M: DNA marker. Lane 1, the piGEM-WRL without restriction endonuclease. Lane 2, the piGEM-WRL was digested by PstI +XhoI. (B) M: DNA marker. Lane 3, the piGEM-RL without restriction endonuclease. Lane 4, the piGEM-RL was digested by PstI +XhoI.

3. We transformed the piGEM-RL into BL21(DE3) and conducted inducible expression. The concentration and color of bacterium liquid are as followed:

Figure 3. The color and the concentration of bacterium liquid. (A) The color degree among different bacterium liquid. The higher concentration of bacterium liquid showed redder. (B) The OD 600 of different concentration of baterium liquid at different time using ultraviolet spectrophotometer.

4. The SDS-PAGE of the cell with the piGEM-RL.

Figure 4. Testing expression of RFP+Laccase in E.coli. M: marker. Lane 1, bacteria untransformed. Lane 2, Bacteria which contain piGEM-RL. Induced at 37centigrades, 180rpm, 10 hours with 0.5mM IPTG.Figure 1.4: We confirmed piGEM-G6X by enzyme digestion using Hind III enzymes in Lane1 and Apal I and Pst I enzymes in Lane2.

In the gel we found that the band located near 80KDa is RFP+Laccase.

5. The activity of laccase：
Using Ultrasonic Cell Disruptor to crush the bacterium in ice-bath. Collect the supernatant and detect the activity of laccase by ABTS method.

Figure 5. The color of supernate and activity of laccase. (A) The higher concentration of laccase showed redder. (B) 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 time.

6. We transformed piGEM-WRL into BL21(DE3) and conducted inducible expression. We got a series of samples which was cultivated at different time and chose one to compare with BL21(DE3) untransformed.

Figure 6. The color of supernate and activity of laccase. (A) The left is BL21(DE3) untransformed and the right is BL21(DE3) transformed with piGEM-WRL. (B) The activity of MamW+RFP+Laccase.

In the end, we compared the two enzyme activity curves.

Figure 7. The comparation of two laccases activity. The red curve represented piGEM-RL and the blue curve presented piGEM-WRL.

This picture showed that the enzyme activity of the laccase coded by piGEM-RL is higher than another. Next we put the RFP+Laccase in the EBFC.

Enzymatic biofuel cell (EBFC)

1. Device 1：EBFC 1.0

Figure 8. The simple EBFC 1.0 made by ourselves.(A) Make this device using discarded bottles with the proton exchange membrane in the middle.(B)Fix the EBFC 1.0 in the foam board, add each component (10 ml in total) into the device according to the protocol. Use multimeter and oscilloscope to test voltage.

Due to the fact that the sealing was not very good and 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.

2. Device 2: EBFC 2.0

Figure 9. The diagram of the EBFC 2.0. Purchase the device from the internet.(A)The middle of the device is proton exchange membrane; the electrode material is carbon paper.(B)The diagram of the fixed EBFC 2.0.

Figure 10. The EBFC 2.0 performances.(A)Before adding enzyme, the voltage was 0V.(B)the scan map of oscilloscope of the left.(C)After adding enzyme, the voltage increased instantly but the voltage was far from stable. After about 5 minutes, the voltage got stable.(D)The voltage reached a stable level of about 160mV and lasted for about 40h. The scan time was 50s and every grid represents 100mV.

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 the small electric current, so we need to further improve the battery.

3. Device 3 EFBC 3.0

Figure 11. The device made by 3D print. Add the components through the upper holes and fix the electrodes to the edge of the holes.

Run the 3D print device and test, it didn’t achieve the desired result; the internal resistance of the battery didn’t get smaller. Apart from lowering 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 gather to the surface of two electrodes and immobilized.

Magnetosome

1. We separated mamAB into 3 parts and amplified each one by common PCR. We subcloned the three parts into pET28a vector successfully, and named piGEM-AB. We verified it using digestion and sequencing.

Figure 12. The construction of piGEM-AB. (A) The three parts of mamAB by PCR using high fidelity DNA polymerase. M: marker. Lane 1, mamAB part1. Lane 2, mamAB part2. Lane 3, mamAB part3.(B)M: marker. Lane 4, digestion the plasmid piGEM-AB by restriction endonuclease ApaI,SapI,NotI.

2. We also amplified mamGFDC+mms6, and mamXY by common PCR. We cloned the two parts into pCDFDuet-1 vector successfully, and named piGEM-G6X. We verified it using digestion and sequencing.

Figure 13. The construction of piGEM-G6X.(A) amplify mamGFDC+mms6 and mamXY by PCR using high fidelity DNA polymerase. M: marker. Lane 1, mamGFDC+mms6. Lane 2, mamXY. (B) M: marker. Lane 3, digestion the plasmid piGEM-G6X by restriction endonuclease HindIII. Lane 4, digestion the plasmid piGEM-G6X by restriction endonuclease ApalI, PstI.

3. We constructed the piGEM-AB and piGEM-G6X successfully and co-transferred them into E.coli, and detected by the colony PCR.

Figure 14. The bacterial colony PCR result. M: marker. Lane 1, the colony with piGEM-AB. Lane 2 and 3, the colony with piGEM-AB+piGEM-G6. Lane 4 and 5, the colony with piGEM-AB+piGEM-G6X.

4. Transmission electron microscope (TEM)

Figure 15. Transmission electron microscopy images of modified E.coli. (A) (B)Images of cells of BL21(DE3) without any vectors prepared on a TEM grid.(C) Images of cells of BL21(DE3) transferred with piGEM-AB.(D) Images of cells of BL21(DE3) co-transferred with piGEM-AB and piGEM-G6. (E)-(H) Images of cells of BL21(DE3) co-transferred with piGEM-AB and piGEM-G6X. Arrows indicate the magnetosome. The scale bar corresponds to 200 nm.

Summary

Table 1. the 16 vectors that we constructed successfully through the three months.

Vectors Inserted gene Gene function Related parts
1 piGEM-AB mamAB Encode a series of protein that is essential for magnetosome synthesis BBa_K1779205
BBa_K1779206
BBa_K1779207
BBa_K1779208
BBa_K1779209
BBa_K1779210
BBa_K1779211
BBa_K1779212
BBa_K1779213
BBa_K1779214
2 piGEM-G6 mamGFDC+mms6 Encode a series of proteins that can regulate the size and shape of crystals in the formation of magnetosome BBa_K1779100
3 piGEM-G6X mamGFDC+mms6+mamXY Encode a series of proteins that can regulate the size and shape of crystals in the formation of magnetosome BBa_K1779100
BBa_K1779101
4 piGEM-R-Lac RFP+laccase Encode a fusion protein which makes laccase visible BBa_K1779204
5 piGEM-W-R-Lac mamW+RFP+laccase Encode a fusion protein to bind laccase to the transmembrane protein MamW, and RFP can make it visible BBa_K1779200
BBa_K1779201
BBa_K1779202
BBa_K1779203
6 piGEM-Plac-H LacI promoter+mamH To make sure whether mamH gene can successfully express
7 piGEM-Plac-G lac promoter+mamG To make sure whether mamG gene can successfully express
8 piGEM-Plac-6 lac promoter+mms6 To make sure whether mamH gene can successfully express
9 piGEM-Plac-Y lac promoter+mamY To make sure whether mamH gene can successfully express
10 piGEM-PH-R-1 mamH promoter+RFP To make sure whether mamH promoter can work
11 piGEM-PG-R mamG promoter+RFP To make sure whether mamG promoter can work
12 piGEM-P6-R mms6 promoter+RFP To make sure whether mms6 promoter can work
13 piGEM-PY-R mamY promoter+RFP To make sure whether mamY promoter can work
14 piGEM-PH-R-2 mamH promoter+RFP To make sure whether mamH promoter can work and verify the backbone of pET28a
15 piGEM-LacI-RFP LacI promoter+RFP To verify the backbone of pET28a
16 piGEM-GFP-PG6-R GFP+mamG-mms6 promoter+RFP To make sure whether mamG-mms6 promoter can work