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Results

Part1: Photosynthetic microorganism system

1.1 Synechocystis sp

1.1.1 Verification of transformation

pCK306 plasmid contains with yellow fluorescent protein. Therefore, to show pCK306 expresses successfully in Synechocystis sp. PCC 6803(cyanobacteria), we use fluorescence microscope to test whether it was transformed into bacteria or not. After cultivating for one week, we use 10μcyanobacteria cultivate dripping on the slides.

Figure 1. Results of yellow fluorescent protein of pCK306 plasmid in Synechocystis PCC6803. L-Rhamose(1g/L) was added and after 36 hours, engineered bacteria showed it expressed successfully.

As we can see from figure 1, yellow fluorescent protein achieves expression, which indicates our transformation method works.

1.1.2 Verification of gene expression

Affinity Chromatography

In addition to knowing transcription of the gene, we want to exhibit the expression of the protein, we insert 6Xhis-tag into lldP and did affinity chromatography to show we finally made it. The result is shown below:

Figure 2a. There were nearly 0.2g bacteria used to testify the expression of lldP.
After affinity chromatography, the protein was electrophoresed through SDS-PAGE. As the red box shows, lldP can be seen . Comparing to wild type, lldP is expressed in Synechocystis

Because of the slow growth of Synechocystis sp. PCC 6803 and time limitation, we did not have too much bacteria, so we only use nearly 0.2g of them to show whether lldP expressed. The color is pale but still can be seen. lldP expression succeeds.

Real-Time Quantitative PCR

To illustrate our project does work. We must prove that our circuit express successfully. Therefore, we did real-time quantitative PCR to demostrate the transcription of the circuit ldhD-lldP, ldhDc-lldP, ldhDnARSdR-lldP and ldhDARSdR-lldP. The result shows as the figure below:

Figure 2b. The figure shows that ldhD-lldP, ldhDc-lldP, ldhDnARSdR-lldP, ldhDARSdR-lldP express successfully.

1.1.3 Detection of Lactate

Finally, we should testify that the whole circuit in Synechocystis sp. PCC 6803(cyanobacteria) works. So we did lactate detection experiment. After adding L-Rhamose(1g/L) for 72h, we use Lactic Acid assay kit, provided by Nanjing Jiancheng Bioengineering, to quantify lactate concentration. The result is shown below, all the cyanobacteria cultivates are converting to OD₇₅₀=1.

Figure 3. Lactate production of engineered Synechocystis sp. PCC6803. Comparison of WT, ldhD-lldP, ldhDc-lldP, ldhDnARSdR-lldP, ldhDARSdR-lldP. Synechocystis grew for 7 days, then L-Rhamose(1g/L) was added. After induced 72 hours, lactate concentration has shown above.

The figure indicates that after transforming our circuit in bacteria, the production and releasing of lactate increase evidently, and ldhDARSdR-lldP is the most efficient one. In summary, our engineered bacteria, Synechocystis sp. PCC 6803(cyanobacteria), do achieve our goal to provide lactate to Shewanella.

1.2 Rhodopseudomonas palustris (Rps)

1.2.1 Verification of gene expression

We transformed pMG105-PpckA-RBS-mleS-TT, pMG105-PpckA-RBS-lldP-TT, pMG105-PpckA-RBS-ldhA-TT, pMG105-PpckA-RBS-mleS-lldP-TT, pMG105-PpckA-RBS-lldP-RBS-ldhA-TT, pMG105-PpckA-RBS-ldhA-RBS-lldP-TT, pMG105-PpckA-RBS-mleS-RBS-lldP-RBS-ldhA-TT into the E.coli BL21. And then, we do Real-Time Quantitative PCR to verificate targeted gene (Figure 4).

(A)

(B)

(C)

Figure 4. Relative expression level of targeted genes in E.coli BL21. We choose mleS(malate dehydrogenase, the conversion of malic acid to L-lactate), lldP(L-lactate permease, the lactate is transported out of the cell) and ldhA(fermentative D-lactate dehydrogenase, NAD-dependent, convert pyruvate to D-lactate) as the reference genes and pMG105 as the standard quantity. (A). The expression level of mleS. (B). The expression level of lldP. (C). The expression level of ldhA. We could find that mleS, lldP and ldhA have a higher expression compared with pMG105. It means every genes can express efficiently.

Figure 4 shows that our genes express successfully on mRNA level.

1.2.2 Detection of Lactate

We detect lactate production of every gene circuits in E.coli BL21 after incubating for 4h (Figure 5).

Figure 5. LB is a kind of medium to incubate bacterial. We detect lactate content of LB and the others. pMG105 has the lowest lactate content because pMG105 doesn’t have any gene to help produce lactate. It proves that all of our gene circuits have ability to produce lactate. Besides, Compared with ldhA and lldP, ldhA-lldP has a better production efficiency. Compared with ldhA-lldP, lldP-ldhA has a better production efficiency. Compared with ldhA-lldP, mleS-lldP has a better production efficiency. Compared with other gene circuits, mleS-lldP-ldhA has a better production efficiency. The result proves our modeling is correct.
The figure 5 shows that our modification is effective. Every gene circuits can help strains produce lactate, and mleS-lldP-ldhA is the most efficient one. Therefore, our construction of gene circuits achieve the goal to help strains produce lactate.

Part2: Electrogenic microorganism system

Shewanella Oneidensis

2.1 Lactate utilization

Since lactate is the carbon source of Shewanella Oneidensis MR-1, overexpression of lactate dehydrogenase might help the bacteria to utilize lactate more efficiently and produce more electricity. We chose dld(encodes D-lactate dehydrogenase) and lldEFG(encodes L-lactate dehydrogenase) to overexpress in Shewanella Oneidensis MR-1.

2.1.1 Gene Expression

To demonstrate that the targeted genes are expressed by engineered Shewanella, we did Real-Time Quantitative PCR.

Figure 6.(A). The expression level of mleS. (B). The epression level of lldP. (C). The expression of ldhA.

As we can see from figure 6, dld could be overexpressed by engineered Shewanella. For further verification, we used the engineered bacteria to produce electricity.

2.1.2 Electrogenesis

By comparing the ability of producing electricity, we might find out whether pYYDT-dld and pYYDT-lldEFG could effectively help Shewanella to produce more electricity.

Figure 7. The comparison of electricity production between engineered Shewanella. (A). Voltage output of WT and recombinant S. oneidensis.

It could be demonstrated that targeted genes could be expressed in the engineered cells. More lactate has been utilized by engineered bacteria, which helps to produce more electricty.

2.2.2 NADH production

NADH is an important part for electrogenesis as it is one of the most important electron carriers in the cell. We hypothesized that more NADH produced by the cell would cause more electricity to be generated. Thus, we chose gapA(encodes glyceraldehyde-3-phosphate dehydrogenase), mdh(encodes NAD-dependent malate dehydrogenase), pflB(encodes pyruvate formate-lyase) and fdh(encodes formate dehydrogenase) to overexpress in Shewanella Oneidensis MR-1.

(1). Gene Expression

To demonstrate that the targeted genes are expressed by engineered Shewanella, we did Real-Time Quantitative PCR.

(A)

(B)

(C)

(D)

(E)

Figure 8. Relative expression level of targeted genes in engineered Shewanella Oneidensis MR-1. We chose gyrB(encodes DNA gyrase B) as the reference genes and 1 as the standard quantity. (A). The expression level of pYYDT-gapA, there was no signal in bacteria which contained pYYDT. (B). The expression level of pYYDT-mdh. (C). The expression level of pYYDT-gapA-mdh. (D). The expression level of pYYDT-pflB. (E). The expression level of pYYDT-pflB-fdh.

As figure 8 shows, gapA, mdh, pflB and fdh could be overexpressed by engineered Shewanella. For further verification, we used the engineered bacteria to produce electricity.

(2). Electrogenesis

By comparing the ability of producing electricity, we might find out whether pYYDT-gapA-mdh and pYYDT-pflB-fdh could effectively help Shewanella to produce more electricity.

Figure 9. The comparison of electricity production between engineered Shewanella. (A). Voltage output of WT and recombinant S. oneidensis.

It could be demonstrated that targeted genes could be expressed in the engineered cells. More NADH has been produced by engineered bacteria, which helps to produce more electricty.

Part3: Whole design

3.1 Pre-experiment of electrogenesis

In the formal experiment. We compared the ability of electricity production between blank medium, Shewanella Oneidensis MR-1 and E.coli to make sure that Shewanella could produce electricity in our device.

Figure 10. The comparison of ability of electricity production between wild type Shewanella Oneidensis MR-1 and E.coli.

We can find that Shewanella could produce electricity and the amount of electricity produced by Shewanella is much more than E.coli.

3.2 Lactate contrast

Then we designed two groups in order to explore the ability of electricity production when we added lactate in Shewanella Oneidensis MR-1.

Figure 11. The influence when adding lactate in wild type Shewanella Oneidensis MR-1.

According to our experiments, we can make a conclusion: compared with medium that doesn’t add lactate, Shewanella oneidensis generates electricity more efficiently in lactate-added medium.

3.3 The difference between carbon rods and carbon cloth

We used carbon rods as electrodes at first. However, we were told by our adviser that the carbon cloth might have better effect for electrogenesis since more bacteria could attach on it.

Figure 12. The comparison of electricity production between using carbon rod and carbon cloth as the electrode.

It is easy to draw the conclusion that the carbon cloth has better effect than carbon rods as electrode.

3.4 The influence of oxygen on electrogenesis

During searching, we found that Shewanella Oneidensis MR-1 had commonly been used in electrogenesis at anaerobic condition. Since we chose Synechocystis PCC6803 as the lactate producer, the oxygen produced by Synechocystis PCC6803 could not be avoided. So we did this group of electrogenesis experiment to find out whether the oxygen would have a negative influence on producing electricity.

Figure 13. The comparison of electricity production between anaerobic circumstance and aerobic circumstance.

Shewanella could produce more electricity at anaerobic circumstance, which means that Synechocystis PCC6803 is not an ideal lactate producer in this electrogenesis system.

3.5 Contrast the symbiotic effect of wild-type strains`

We tried to compare the effects of Synechocystis PCC6803 and Rhodopseudomonas palustris by constructing a MFC system.

Figure 14. The comparison of electricity production between Synechocystis PCC6803 and Rhodopseudomonas palustris

The figure shows that the effect of Rhodopseudomonas palustris is better than Synechocystis PCC6803. We believe that the oxygen produced by Synechocystis PCC6803 mainly reduces the electricity production effect.

3.6 Functional verification of engineered Synechocystis PCC6803

Figure 15. The comparison of electricity production between wild type and engineered Synechocystis PCC6803

In this figure we can find that the engineered Synechocystis PCC6803 shows its function in MFC system.

College of Life Science & Technology
Huazhong University of Science and Technology
Add: 1037 Luoyu Road, Wuhan, Hubei, China; P.C: 430074
Copyright ©2018 Huazhong University of Science & Technology. Produced By HUST-China

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+		            <span dir="auto">Team:HUST-China/Results</span>
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                                <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Modeling overview">Modeling overview</a></li>
-                               <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Comparison between PSB">Comparison between PSB</a></li>
+                               <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/model_of_systems">Model of systems</a></li>
                                <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Sort of three genes">Sort of three genes</a></li>
-                               <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Software">Intelligent device software</a></li>
+                               <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Software">Software</a></li>
                            </ul>
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                            <a href="#" data-toggle="dropdown" class="dropdown-toggle waves-effect waves-dark">HP<b class="caret"></b></a>
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-                               <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Collaborations">Human Practices</a></li>
+                               <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Human Practices">Human Practices</a></li>
-                               <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Education Engagement">Education&Engagement</a></li>
+                               <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Education_Engagement">Education&amp;Engagement</a></li>
                            </ul>
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                                <li><a class="waves-effect waves-dark" href="https://2018.igem.org/Team:HUST-China/Attributions">Attributions</a></li>
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                        <div class="info-blocks-in" style="background-color: #ffffff; border: 1px solid #eeeeee;border-radius:5px;">
                                <h3><strong><span class="red-content">1.1 Synechocystis sp</span></strong></h3>
-                               <h3>1.1.1 Verification of transformation</h3>
+                               <h3><b>1.1.1 Verification of transformation</b></h3>
                                <p>pCK306 plasmid contains with yellow fluorescent protein. Therefore, to show pCK306 expresses successfully in Synechocystis sp. PCC 6803(cyanobacteria), we use fluorescence microscope to test whether it was transformed into bacteria or not. After cultivating for one week, we use 10μcyanobacteria cultivate dripping on the slides.</p>
                              <div class="col-md-8 col-md-offset-1" style="text-align: center;">
                                      <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/c/cf/T--HUST-China--2018-result-YFP.png">
-                                     <p>Figure 1. Results of yellow fluorescent protein of pCK306 plasmid in Synechocystis PCC6803. L-Rhamose(1g/L) was added and after 36 hours, engineered bacteria showed it expressed successfully.</p>
+                                     <p>Figure 1. Results of yellow fluorescent protein of pCK306 plasmid in Synechocystis PCC6803. L-Rhamose(1g/L) was added and after 36 hours, engineered bacteria showed it expressed successfully.</i></p>
                              </div>
                              <div class="col-md-12">
-                                 <p>As we can see from figure 1, yellow fluorescent protein achieves expression, which indicates our transformation method works.</p>
+                                 <p>As we can see from figure 1, yellow fluorescent protein achieves expression, which indicates our transformation method works.</p><br/>
                              </div>
                            <div class="col-md-12">
-                             <h3>1.1.2 Verification of transformation </h3>
+                             <h3><b>1.1.2 Verification of gene expression </b></h3>
                                <p><strong class="red-content">Affinity Chromatography</strong></p>
                                <p>In addition to knowing transcription of the gene, we want to exhibit the expression of the protein, we insert 6Xhis-tag into lldP and did affinity chromatography to show we finally made it. The result is shown below:</p>
@@ Line 253: / Line 386: @@
                            <div class="col-md-8 col-md-offset-1" style="text-align: center;">
                                      <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/6/68/T--HUST-China--2018-result-lldP.png">
-                                     <p>Figure 3b. There were nearly 0.2g bacteria used to testify the expression of lldP. After affinity chromatography, the protein was electrophoresed through SDS-PAGE. As the red box shows, lldP can be seen . Comparing to wild type, lldP is expressed in Synechocystis</p>
+                                    <div class="col-md-offset-1">
+                                     <p style="font-size: 5px;">Figure 2a. There were nearly 0.2g bacteria used to testify the expression of lldP.</i><br> After affinity chromatography, the protein was electrophoresed through SDS-PAGE. As the red box shows, lldP can be seen . Comparing to wild type, lldP is expressed in Synechocystis</p>
+                          </div>
                            </div>
                            <div class="col-md-12">
-                                 <p>Because of the slow growth of Synechocystis sp. PCC 6803 and time limitation, we did not have too much bacteria, so we only use nearly 0.2g of them to show whether lldP expressed. The color is pale but still can be seen. lldP expression succeeds.</p>
+                                 <p>Because of the slow growth of Synechocystis sp. PCC 6803 and time limitation, we did not have too much bacteria, so we only use nearly 0.2g of them to show whether lldP expressed. The color is pale but still can be seen. lldP expression succeeds.</p><br/>
                            </div>
                            <div class="col-md-12">
-                             <h3>1.1.3 Detection of Lactate</h3>
+                              <p><strong class="red-content">Real-Time Quantitative PCR</strong></p>
+                              <p>To illustrate our project does work. We must prove that our circuit express successfully. Therefore, we did real-time quantitative PCR to demostrate the transcription of the circuit ldhD-lldP, ldhDc-lldP, ldhDnARSdR-lldP and ldhDARSdR-lldP. The result shows as the figure below:</p>
+                          </div>
+                          <div class="col-md-8 col-md-offset-1" style="text-align: center;">
+ <img class="img-responsive" height="400" width="300" src="https://static.igem.org/mediawiki/2018/2/20/T--HUST-China--2018-result_xiaoziyang_hong.png">
+ <img class="img-responsive"  height="400" width="300" src="https://static.igem.org/mediawiki/2018/a/ab/T--HUST-China--2018-result_xiaoziyang_zi.png">
+ <img class="img-responsive"  height="400" width="300" src="https://static.igem.org/mediawiki/2018/9/96/T--HUST-China--2018-result_xiaoziyang_lan.png">
+ <img class="img-responsive"  height="400" width="300" src="https://static.igem.org/mediawiki/2018/8/8d/T--HUST-China--2018-result_xiaoziyang_ju.png">
+                                    <div class="col-md-offset-1">
+                                    <p style="font-size: 5px;">Figure 2b. The figure shows that ldhD-lldP, ldhDc-lldP, ldhDnARSdR-lldP, ldhDARSdR-lldP express successfully.</p>
+                          </div>
+                          </div>
+                          <div class="col-md-12">
+                          </div>
+                          <div class="col-md-12">
+                             <h3><b>1.1.3 Detection of Lactate</b></h3>
                                <p>Finally, we should testify that the whole circuit in Synechocystis sp. PCC 6803(cyanobacteria) works. So we did lactate detection experiment. After adding L-Rhamose(1g/L) for 72h, we use Lactic Acid assay kit, provided by Nanjing Jiancheng Bioengineering, to quantify lactate concentration. The result is shown below, all the cyanobacteria cultivates are converting to OD<sub>750</sub>=1.</p>
-                         </div>
+                                </div>
-                         <div class="col-md-8 col-md-offset-1" style="text-align: center;">
+                                <div  class="col-md-5 col-md-offset-3" > <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/d/d6/T--HUST-China--2018-lactate_%281%29.png"></div>
-                                    <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/8/8e/T--HUST-China--2018-result-003.png">
+                                 <div class="col-md-12 col-md-offset-1">
-                                    <p>Figure 3.  Lactate production of engineered Synechocystis PCC6803. Comparison of WT, ldhD-lldP, ldhDc-lldP, ldhDnARSdR-lldP, ldhDARSdR-lldP. Synechocystis grew for 7 days(OD=1), then L-Rhamose(1g/L) was added. After induced 72 hours, lactate concentration in culture supernatant has shown above.</p>
+                             <p style="font-size: 5px;" >Figure 3. <i> Lactate production of engineered Synechocystis sp. PCC6803. </i>Comparison of WT, ldhD-lldP, ldhDc-lldP, ldhDnARSdR-lldP, ldhDARSdR-lldP. Synechocystis grew for 7 days, then L-Rhamose(1g/L) was added. After induced 72 hours, lactate concentration has shown above.</p>
-                         </div>
+                            </div>
                            <div class="col-md-12">
                                <p>The figure indicates that after transforming our circuit in bacteria, the production and releasing of lactate increase evidently, and ldhDARSdR-lldP is the most efficient one. In summary, our engineered bacteria, Synechocystis sp. PCC 6803(cyanobacteria), do achieve our goal to provide lactate to Shewanella.</p>
@@ Line 278: / Line 433: @@
                  <div class="info-blocks-in" style="background-color: #ffffff; border: 1px solid #eeeeee;border-radius:5px;">
                          <h3><strong><span class="red-content">1.2 Rhodopseudomonas palustris (Rps)</span></strong></h3>
-                         <h3>1.2.1 Verification of gene expression</h3>
+                         <h3><b>1.2.1 Verification of gene expression<b/></h3>
-                         <p>We transformed pMG105-PpckA-RBS-mleS-TT, pMG105-PpckA-RBS-lldP-TT, pMG105-PpckA-RBS-ldhA-TT, pMG105-PpckA-RBS-mleS-lldP-TT, pMG105-PpckA-RBS-lldP-RBS-ldhA-TT, pMG105-PpckA-RBS-ldhA-RBS-lldP-TT, pMG105-PpckA-RBS-mleS-RBS-lldP-RBS-ldhA-TT into the E.coli BL21. And then, we do Real-Time Quantitative PCR to verificate targeted gene (Figure 1).</p>
+                         <p>We transformed pMG105-PpckA-RBS-mleS-TT, pMG105-PpckA-RBS-lldP-TT, pMG105-PpckA-RBS-ldhA-TT, pMG105-PpckA-RBS-mleS-lldP-TT, pMG105-PpckA-RBS-lldP-RBS-ldhA-TT, pMG105-PpckA-RBS-ldhA-RBS-lldP-TT, pMG105-PpckA-RBS-mleS-RBS-lldP-RBS-ldhA-TT into the E.coli BL21. And then, we do Real-Time Quantitative PCR to verificate targeted gene (Figure 4).</p>
                          <div class="col-md-12" style="text-align: center;">
                                <div class="col-md-4">
                                      <p>(A)</p>
-                                     <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/d/d2/T--HUST-China--2018-result-zhaozehong-a.png">
+                                     <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/archive/2/20/20181017212558%21T--HUST-China--2018-result-pic1.png" style="width: 590px;height:490px">
                                </div>
                                <div class="col-md-4">
                                      <p>(B)</p>
-                                     <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/3/35/T--HUST-China--2018-result-zhaozehong-b.png">
+                                     <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/archive/2/20/20181017212625%21T--HUST-China--2018-result-pic1.png" style="width: 590px;height:490px">
                                </div>
                                <div class="col-md-4">
                                      <p>(C)</p>
-                                     <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/1/1f/T--HUST-China--2018-result-zhaozehong-c.png">
+                                     <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/2/20/T--HUST-China--2018-result-pic1.png" style="width: 590px;height:490px">
                                </div>
-                                <p>Figure 1：(A). The expression level of mleS. (B). The epression level of lldP. (C). The expression of ldhA.</p>
+                                <p>Figure 4. Relative expression level of targeted genes in E.coli BL21. We choose mleS(malate dehydrogenase, the conversion of malic acid to L-lactate), lldP(L-lactate permease, the lactate is transported out of the cell) and ldhA(fermentative D-lactate dehydrogenase, NAD-dependent, convert pyruvate to D-lactate) as the reference genes and pMG105 as the standard quantity. (A). The expression level of mleS. (B). The expression level of lldP. (C). The expression level of ldhA. We could find that mleS, lldP and ldhA have a higher expression compared with pMG105. It means every genes can express efficiently.</p></i>
                           </div>
                          <div class="col-md-12">
-                                 <p>Figure 1 shows that our genes express successfully on mRNA level. </p>
+                                 <p>Figure 4 shows that our genes express successfully on mRNA level. </p>
                          </div>
-                         <div class="col-md-12">
+                         <div class="col-md-12"><br/>
-                             <h3>1.2.2 Detection of Lactate</h3>
+                             <h3><b>1.2.2 Detection of Lactate</b></h3>
-                               <p>We detect lactate production of every gene circuits in E.coli BL21 after incubating for 4h  (Figure 2). </p>
+                               <p>We detect lactate production of every gene circuits in E.coli BL21 after incubating for 4h  (Figure 5). </p>
                           </div>
                           <div class="col-md-8 col-md-offset-1" style="text-align: center;">
-                                     <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/0/02/T--HUST-China--2018-result-rusuan01.png">
+                                     <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/a/af/T--HUST-China--2018-result-rusuan04.png">
-                                     <p>Figure 2 shows that our modification is effective. Every gene circuits can help strains produce lactate, and mleS-lldP-ldhA is the most efficient one. Therefore, our construction of gene circuits achieve the goal to help strains produce lactate.  </p>
+                                     <p>Figure 5. LB is a kind of medium to incubate bacterial. We detect lactate content of LB and the others. pMG105 has the lowest lactate content because pMG105 doesn’t have any gene to help produce lactate. It proves that all of our gene circuits have ability to produce lactate. Besides, Compared with ldhA and lldP, ldhA-lldP has a better production efficiency. Compared with ldhA-lldP, lldP-ldhA has a better production efficiency. Compared with ldhA-lldP, mleS-lldP has a better production efficiency. Compared with other gene circuits, mleS-lldP-ldhA has a better production efficiency. The result proves our modeling is correct.   <br> The figure 5 shows that our modification is effective. Every gene circuits can help strains produce lactate, and mleS-lldP-ldhA is the most efficient one. Therefore, our construction of gene circuits achieve the goal to help strains produce lactate.  </p>
                           </div>
                           <!-------------------------------------------------------------------------->
@@ Line 341: / Line 496: @@
                          <div class="col-md-8 col-md-offset-1" style="text-align: center;">
                                      <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/a/a1/T--HUST-China--2018-result-fig1.png">
-                                     <p>Figure 1：(A). The expression level of mleS. (B). The epression level of lldP. (C). The expression of ldhA.</p>
+                                     <p>Figure 6.(A). The expression level of mleS. (B). The epression level of lldP. (C). The expression of ldhA.</p></i>
                           </div>
                              <div class="col-md-12">
-                                 <p>As we can see from figure 1, dld could be overexpressed by engineered Shewanella. For further verification, we used the engineered bacteria to produce electricity.</p>
+                                 <p>As we can see from figure 6, dld could be overexpressed by engineered Shewanella. For further verification, we used the engineered bacteria to produce electricity.</p>
                              </div>
-                             <div class="col-md-12">
+                             <div class="col-md-12"><br/>
                                  <h3>2.1.2 Electrogenesis</h3>
                                  <p>By comparing the ability of producing electricity, we might find out whether pYYDT-dld and pYYDT-lldEFG could effectively help Shewanella to produce more electricity. </p>
@@ Line 355: / Line 510: @@
                              <div class="col-md-8 col-md-offset-1" style="text-align: center;">
                                      <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/5/5d/T--HUST-China--2018-result-fig2.png">
-                                     <p>Figure 2.The comparison of electricity production between engineered Shewanella. (A). Voltage output of WT and recombinant S. oneidensis.</p>
+                                     <p>Figure 7. The comparison of electricity production between engineered Shewanella. (A). Voltage output of WT and recombinant S. oneidensis.</p></i>
                              </div>
@@ Line 362: / Line 517: @@
                              </div>
-                              <div class="col-md-12">
+                              <div class="col-md-12"><br/>
                                  <h3><strong>2.2.2 NADH production</strong></h3>
                                  <p>NADH is an important part for electrogenesis as it is one of the most important electron carriers in the cell. We hypothesized that more NADH produced by the cell would cause more electricity to be generated. Thus, we chose gapA(encodes glyceraldehyde-3-phosphate dehydrogenase), mdh(encodes NAD-dependent malate dehydrogenase), pflB(encodes pyruvate formate-lyase) and fdh(encodes formate dehydrogenase) to overexpress in Shewanella Oneidensis MR-1.</p>
@@ Line 391: / Line 546: @@
                                    </div>
                                     <div class="col-md-12">
-                                        <p>Figure 3. Relative expression level of targeted genes in engineered Shewanella Oneidensis MR-1. We chose gyrB(encodes DNA gyrase B) as the reference genes and 1 as the standard quantity. (A). The expression level of pYYDT-gapA, there was no signal in bacteria which contained pYYDT. (B). The expression level of pYYDT-mdh. (C). The expression level of pYYDT-gapA-mdh. (D). The expression level of pYYDT-pflB. (E). The expression level of pYYDT-pflB-fdh.</p>
+                                        <p>Figure 8. Relative expression level of targeted genes in engineered Shewanella Oneidensis MR-1.</i> We chose gyrB(encodes DNA gyrase B) as the reference genes and 1 as the standard quantity. (A). The expression level of pYYDT-gapA, there was no signal in bacteria which contained pYYDT. (B). The expression level of pYYDT-mdh. (C). The expression level of pYYDT-gapA-mdh. (D). The expression level of pYYDT-pflB. (E). The expression level of pYYDT-pflB-fdh.</p></i>
                                    </div>
                                  </div>
                                  <div class="col-md-12">
-                                     <p>As figure 3 shows, gapA, mdh, pflB and fdh could be overexpressed by engineered Shewanella. For further verification, we used the engineered bacteria to produce electricity.</p>
+                                     <p>As figure 8 shows, gapA, mdh, pflB and fdh could be overexpressed by engineered Shewanella. For further verification, we used the engineered bacteria to produce electricity.</p>
                                  </div>
                                  <div class="col-md-12">
@@ Line 403: / Line 558: @@
                                  <div class="col-md-8 col-md-offset-1" style="text-align: center;">
                                      <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/0/0d/T--HUST-China--2018-result-fig8.png">
-                                     <p>Figure 4. The comparison of electricity production between engineered Shewanella. (A). Voltage output of WT and recombinant S. oneidensis. </p>
+                                     <p>Figure 9. The comparison of electricity production between engineered Shewanella. (A). Voltage output of WT and recombinant S. oneidensis. </p></i>
                                  </div>
                                  <div class="col-md-12">
@@ Line 417: / Line 572: @@
              <div class="col-md-12 content-text">
                  <div class="about-logo">
-                  <h3><strong>Part3: <span class="red-content">Whole design</span></strong></h3>
+                 <h3><strong>Part3: <span class="red-content">Whole design</span></strong></h3>
                  </div>
              </div>
@@ Line 426: / Line 581: @@
                  <i class="icon-info-blocks material-icons hidden-xs"><img class="img-responsive" src="https://static.igem.org/mediawiki/2018/9/98/T--HUST-China--2018-coin17.png"></i>
                        <div class="info-blocks-in" style="background-color: #ffffff; border: 1px solid #eeeeee;border-radius:5px;">
-                            <div class="col-md-12">
+<div class="col-md-12">
-                                 <h3>3.1 Contrast the symbiotic effect of wild-type strains`</h3>
+                                 <h3>3.1  Pre-experiment of electrogenesis </h3>
-                                 <p>We tried to compare the effects of Synechocystis PCC6803 and Rhodopseudomonas palustris by constructing a MFC system.</p>
+                                 <p>In the formal experiment. We compared the ability of electricity production between blank medium, Shewanella Oneidensis MR-1 and E.coli to make sure that Shewanella could produce electricity in our device.</p>
                              </div>
                              <div class="col-md-8 col-md-offset-1" style="text-align: center;">
-                                     <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/c/cc/T--HUST-China--2018-result-xiwachandian01.png">
+                                     <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/4/44/T--HUST-China--2018-pre_experienment.png">
-                                    <p>Figure 1. The comparison of electricity production between Synechocystis PCC6803 and Rhodopseudomonas palustris</p>
+                                     <p>Figure 10. The comparison of ability of electricity production between wild type Shewanella Oneidensis MR-1 and E.coli.</p></i>
                              </div>
                              <div class="col-md-12">
-                                 <p>The figure shows that the effect of Rhodopseudomonas palustris is better than Synechocystis PCC6803. We believe that the oxygen produced by Synechocystis PCC6803 mainly reduces the electricity production effect.</p>
+                                 <p>We can find that Shewanella could produce electricity and the amount of electricity produced by Shewanella is much more than E.coli.</p>
                              </div>
-                            <!--------------------------------------------------->
+ <!--------------------------------------------------->
-                            <div class="col-md-12">
+                          <div class="col-md-12"><br/>
-                                 <h3>3.2 Functional verification of engineered Synechocystis PCC6803</h3>
+                                 <h3>3.2 Lactate contrast</h3>
+                                <p>Then we designed two groups in order to explore the ability of electricity production when we added lactate in Shewanella Oneidensis MR-1.</p>
                              </div>
                              <div class="col-md-8 col-md-offset-1" style="text-align: center;">
-                                     <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/2/22/T--HUST-China--2018-result-xiwachandian02.png">
+                                     <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/d/d7/T--HUST-China--2018-result_kk_figure_4_1.png">
-                                      <p>Figure 2. The comparison of electricity production between wild type and engineered Synechocystis PCC6803</p>
+                                      <p>Figure 11. The influence when adding lactate in wild type Shewanella Oneidensis MR-1.</p></i>
                              </div>
                              <div class="col-md-12">
-                                 <p>In this figure we can find that the engineered Synechocystis PCC6803 shows its function in MFC system.</p>
+                                 <p>According to our experiments, we can make a conclusion: compared with medium that doesn’t add lactate, Shewanella oneidensis generates electricity more efficiently in lactate-added medium.</p>
                              </div>
+ <div class="col-md-12">
+                                 <h3>3.3 The difference between carbon rods and carbon cloth</h3>
-                            <!--------------------------------------------------->
-                            <div class="col-md-12">
-                                 <h3>3. The difference between carbon rods and carbon cloth</h3>
                                  <p>We used carbon rods as electrodes at first. However, we were told by our adviser that the carbon cloth might have better effect for electrogenesis since more bacteria could attach on it.</p>
                              </div>
@@ Line 463: / Line 616: @@
                              <div class="col-md-8 col-md-offset-1" style="text-align: center;">
                                      <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/2/25/T--HUST-China--2018-result-xiwachandian03.png">
-                                      <p>Figure 4. The comparison of electricity production between using carbon rod and carbon cloth as the electrode.</p>
+                                      <p>Figure 12. The comparison of electricity production between using carbon rod and carbon cloth as the electrode.</p></i>
                              </div>
@@ Line 471: / Line 624: @@
                              <!--------------------------------------------------->
-                            <div class="col-md-12">
+                             <div class="col-md-12"><br/>
                                  <h3>3.4 The influence of oxygen on electrogenesis</h3>
                                  <p>During searching, we found that Shewanella Oneidensis MR-1 had commonly been used in electrogenesis at anaerobic condition. Since we chose Synechocystis PCC6803 as the lactate producer, the oxygen produced by Synechocystis PCC6803 could not be avoided. So we did this group of electrogenesis experiment to find out whether the oxygen would have a negative influence on producing electricity.</p>
@@ Line 477: / Line 631: @@
                              <div class="col-md-8 col-md-offset-1" style="text-align: center;">
-                                     <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/1/18/T--HUST-China--2018-result-xiwachandian04.png">
+                                     <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/f/f5/T--HUST-China--2018-%E6%9C%89%E6%B0%A7%E6%97%A0%E6%B0%A7.png">
-                                      <p>Figure 5. The comparison of electricity production between anaerobic circumstance and aerobic circumstance.</p>
+                                      <p>Figure 13. The comparison of electricity production between anaerobic circumstance and aerobic circumstance.</p></i>
                              </div>
@@ Line 485: / Line 639: @@
                              </div>
+                            <!--------------------------------------------------->
+                            <div class="col-md-12"><br/>
+                                <h3>3.5 Contrast the symbiotic effect of wild-type strains`</h3>
+                                <p>We tried to compare the effects of Synechocystis PCC6803 and Rhodopseudomonas palustris by constructing a MFC system.</p>
+                            </div>
+                            <div class="col-md-8 col-md-offset-1" style="text-align: center;">
+                                    <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/c/cc/T--HUST-China--2018-result-xiwachandian01.png">
+                                    <p>Figure 14. The comparison of electricity production between Synechocystis PCC6803 and Rhodopseudomonas palustris</p></i>
+                            </div>
+                            <div class="col-md-12">
+                                <p>The figure shows that the effect of Rhodopseudomonas palustris is better than Synechocystis PCC6803. We believe that the oxygen produced by Synechocystis PCC6803 mainly reduces the electricity production effect.</p>
+                            </div>
+                            <!--------------------------------------------------->
+ <div class="col-md-12"><br/>
+                                <h3>3.6 Functional verification of engineered Synechocystis PCC6803</h3>
+                            </div>
+                            <div class="col-md-8 col-md-offset-1" style="text-align: center;">
+                                    <img class="img-responsive" src="https://static.igem.org/mediawiki/2018/2/22/T--HUST-China--2018-result-xiwachandian02.png">
+                                     <p>Figure 15. The comparison of electricity production between wild type and engineered Synechocystis PCC6803</p></i>
+                            </div>
+                            <div class="col-md-12">
+                                <p>In this figure we can find that the engineered Synechocystis PCC6803 shows its function in MFC system.</p>
+                            </div>
                        </div>
@@ Line 501: / Line 684: @@
            <div class="copyright col-md-12" style="text-align: center;color: gray; font-size: 14px;">
                <span>
-                 College of Life Science & Technology<br>
+                 College of Life Science &amp; Technology<br>
                  Huazhong University of Science and Technology<br>
                  Add: 1037 Luoyu Road, Wuhan, Hubei, China; P.C: 430074<br>
-                 Copyright &copy;2018 Huazhong University of Science & Technology. Produced By </span><a href="https://2018.igem.org/Team:HUST-China" target="_blank" style="color:white;">HUST-China</a>
+                 Copyright &#169;2018 Huazhong University of Science &amp; Technology. Produced By </span><a href="https://2018.igem.org/Team:HUST-China" target="_blank" style="color:white;">HUST-China</a>
            </div>
        </div>
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Difference between revisions of "Team:HUST-China/Results"

Latest revision as of 03:59, 18 October 2018

Team:HUST-China/Results

Team:HUST-China/Results

Results

Part1: Photosynthetic microorganism system

1.1 Synechocystis sp

1.1.1 Verification of transformation

1.1.2 Verification of gene expression

1.1.3 Detection of Lactate

1.2 Rhodopseudomonas palustris (Rps)

1.2.1 Verification of gene expression

1.2.2 Detection of Lactate

Part2: Electrogenic microorganism system

Shewanella Oneidensis

2.1 Lactate utilization

2.1.1 Gene Expression

2.1.2 Electrogenesis

2.2.2 NADH production

Part3: Whole design

3.1 Pre-experiment of electrogenesis

3.2 Lactate contrast

3.3 The difference between carbon rods and carbon cloth

3.4 The influence of oxygen on electrogenesis

3.5 Contrast the symbiotic effect of wild-type strains`

3.6 Functional verification of engineered Synechocystis PCC6803