Difference between revisions of "Team:KUAS Korea/Results"

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<p><font size=4>All the graphs are measured voltages of our bio-batteries. The voltages were measured by  Keithley Digital Multimeter. The unit of X axis is hours measured.</font></p><br><br>
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<p><font size=4>Our project is to make circuits that express chromoproteins when heme is present. To do this, we used a mechanism in which bacteria respond to heme to make an efflux pump. In this system, HssS and HssR were used to recognize from the outside of the cell and HrtR to recognize from the inside.<font></p><br><br>
  
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<h4>1. MFC Validation</h4><br>
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<h4>1. HrtR System</h4><br>
  
  
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<p><font size=4>We measured the voltage that <em>Shewanella oneidensis</em> MR-1 produce when formate is given since MR-1 uses formate to generate electricity. As controls, we measured the voltage of the battery with <em>E. coli</em> BW25113 in the anode chamber, and the battery without microbes. Methylene blue was given as the mediator. The purpose of this experiment is to make sure that our battery device works as an MFC. </font></p><br>
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<p><font size=4>HrtR is a self-regulator that binds directly to the promoter’s palindrome sequence(ATGACACAGTGTCAT) in front of its CDS. When it binds to the promoter, it acts as an inhibitor that prevents the transcription of itself.</font></p><br>
 
                        
 
                        
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                         <img src=" https://static.igem.org/mediawiki/2016/e/ef/Korea_U_Seoul_MFC.jpeg " width=100%>
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<p><font size=4>As you can see in the graph above, the battery with <em>shewanella oneidensis</em> MR-1 generated electricity while the controls did not. This indicates that out battery device works as an MFC.</font></p>
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<p><font size=4>We ordered the hrtR sequence of the gram-positive <em>Lactococcus lactis</em> in IDT and cloned it into DH5alpha using pSB1C3 and TA cloning. In the picture above, the white colonies were inserted with the HrtR and the RFP was deleted. The red one is that the RFP still exists because HrtR is not inserted normally. </font></p>
 
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<h4>2. EFC Validation</h4><br>
 
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<p><font size=4>Diaphorase expressed <em>E. coli</em> BL21(DE3) was lysed by sonication and was applied into the anode chamber of our battery device. Methylene blue was given as the mediator. Lysed BL21(DE3) with void vector was the control. NADH was added to both batteries.          </font></p>          <br> 
 
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                        <img src="https://static.igem.org/mediawiki/2016/6/65/Korea_U_Seoul_EFC.jpeg" width=100%>
 
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<h4>2. Chromoprotein</h4><br>
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<p><font size=4>Commonly used GFPs must be exposed to ultraviolet light in order to see strong green color. So we planned to use chromoprotein instead of fluorescent protein because we thought that fluorescent proteins would be difficult to use in common life. The 2013 Uppsala team's project was using <em>Lactobacillus</em> and chromoprotein, and we decided to use these parts. This photograph shows the expression of their amilCP in <em>E. coli</em> DH5alpha. AmilCP will be more noticeable because it has a blue color that is complementary to the color of the feces.</font></p><br> 
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<p><font size=4>As you can see in the graph above, the battery with diaphorase showed higher voltages. This indicates that the diaphorase produced electricity. However, the voltage differences are very small. This could be due to the state of diaphorase, which was not fixed onto the electrode. Also it could be due to the components that exist in crude bacteria extract since there are many reducing or oxidizing agents in bacterial cells.</font> </p><br><br>
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<p><font size=4>Our final design using above system is as follows. HrtR is expressed in cells at a constant concentration by negative feedback. When HtrR binds to heme, its inhibitor function is lost and transcription occurs. Therefore, the concentration of chromoprotein increases and the color of the bacteria in the feces becomes darker.</font> </p><br><br>
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<h4>3. EMFC Operation</h4><br>
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<p><font size=4>Our final goal was to prove that our device works as an EMFC. <em>Shewanella oneidensis</em> MR-1, BW25113 with displayed agar degrading enzymes, cell lysate of diaphorase expressed BL21(DE3), and cell lysate of TEV expressed BL21(DE3) was put into the anode chamber. Agar was used as the substrate. The battery device without agar was set as the control.  </font></p>    <br><br>           
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                        <img src=" https://static.igem.org/mediawiki/2016/e/eb/Korea_U_Seoul_EMFC.jpeg" width=100%>
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<p><font size=4>As you can see in the graph above, the battery with agar generated more electricity than the control. Our prototype EMFC apparently works as expected. </font></p>
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<br><p><font size=4>However, the voltage generated is not very high. This was expected due to some reasons. First, our battery device was not designed to generated high electricity. To get high yield, you need electrodes with large surface area but the electrodes of our device has small surface area since it is only a thin carbon paper with coated back. We designed our device this way for precise comparisont. Second, our added diaphorase was in free state which means the majority of the diaphorase was not doing much to generate electricity. A lot of EFC relate theses fixes diaphorase onto the electrode for higher yield, and stabilization of the enzymes. </font></p>
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<br><p><font size=4>Since we succeeded in operating the prototype EMFC we designed, our next goal is to improve electricity yield. This includes improving the battery device with better electrodes, optimizing the amount of reagents and cells in the device, and purifying enzymes to get rid of uselss cell lysate.</font> </p>
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Revision as of 14:15, 1 November 2017

Results

Results


Our project is to make circuits that express chromoproteins when heme is present. To do this, we used a mechanism in which bacteria respond to heme to make an efflux pump. In this system, HssS and HssR were used to recognize from the outside of the cell and HrtR to recognize from the inside.



1. HrtR System


HrtR is a self-regulator that binds directly to the promoter’s palindrome sequence(ATGACACAGTGTCAT) in front of its CDS. When it binds to the promoter, it acts as an inhibitor that prevents the transcription of itself.





We ordered the hrtR sequence of the gram-positive Lactococcus lactis in IDT and cloned it into DH5alpha using pSB1C3 and TA cloning. In the picture above, the white colonies were inserted with the HrtR and the RFP was deleted. The red one is that the RFP still exists because HrtR is not inserted normally.



2. Chromoprotein


Commonly used GFPs must be exposed to ultraviolet light in order to see strong green color. So we planned to use chromoprotein instead of fluorescent protein because we thought that fluorescent proteins would be difficult to use in common life. The 2013 Uppsala team's project was using Lactobacillus and chromoprotein, and we decided to use these parts. This photograph shows the expression of their amilCP in E. coli DH5alpha. AmilCP will be more noticeable because it has a blue color that is complementary to the color of the feces.





Our final design using above system is as follows. HrtR is expressed in cells at a constant concentration by negative feedback. When HtrR binds to heme, its inhibitor function is lost and transcription occurs. Therefore, the concentration of chromoprotein increases and the color of the bacteria in the feces becomes darker.