Difference between revisions of "Team:Korea U Seoul/Proof"
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| + | <h3>Proof of Concept</h3><Br><Br> | ||
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| + | <p><font size=4>One of our parts, diaphorase has been cloned into pB3 vector, which has T7 promoter, and was expressed in BL21(DE3).<br> | ||
| + | <div class="image image-full"><img src="https://static.igem.org/mediawiki/2016/a/ac/Korea_U_Seoul_Proof_pB3.jpg"></div> | ||
| + | Diaphorase assay was done using DCPIP, and diaphorase was also tested in our own battery device to check whether it can be used to generate electricity.</font></p> | ||
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<h4>1. Diaphorase assay</h4> | <h4>1. Diaphorase assay</h4> | ||
| − | <p>DCPIP is a redox dye, which is blue when oxidized and colourless when reduced. It can be used to check whether diaphorase has activity since it can be reduced by diaphorase.</p> | + | <p><font size=4>DCPIP is a redox dye, which is blue when oxidized and colourless when reduced. It can be used to check whether diaphorase has activity since it can be reduced by diaphorase.</font></p><br> |
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| − | <img src="https://static.igem.org/mediawiki/2016/2/29/Korea_U_Seoul_diaphorase.jpeg"> | + | <img src="https://static.igem.org/mediawiki/2016/2/29/Korea_U_Seoul_diaphorase.jpeg" width=100%><br><br><br> |
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| + | <p><font size=4>We cultured BL21(DE3) with pB3_diaphorase and BL21(DE3) with pB3 void vector. <br> | ||
| + | Both were induced with IPTG, and were purified using NI-NTA Spin Kit from Quiagen.<br> | ||
| + | Purified cell extract with diaphorase became colourless as we added NADH. However, the colour of purified cell extract without diaphorase did not change. <br> | ||
| + | We confirmed that our diaphorase has its activity. </font></p><br> | ||
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| + | <h4>2. EFC validation</h4><br> | ||
| + | <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><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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Latest revision as of 16:31, 18 October 2016
Proof of Concept
Proof of concept
One of our parts, diaphorase has been cloned into pB3 vector, which has T7 promoter, and was expressed in BL21(DE3).
Diaphorase assay was done using DCPIP, and diaphorase was also tested in our own battery device to check whether it can be used to generate electricity.
1. Diaphorase assay
DCPIP is a redox dye, which is blue when oxidized and colourless when reduced. It can be used to check whether diaphorase has activity since it can be reduced by diaphorase.
We cultured BL21(DE3) with pB3_diaphorase and BL21(DE3) with pB3 void vector.
Both were induced with IPTG, and were purified using NI-NTA Spin Kit from Quiagen.
Purified cell extract with diaphorase became colourless as we added NADH. However, the colour of purified cell extract without diaphorase did not change.
We confirmed that our diaphorase has its activity.
2. EFC validation
Diaphorase expressed E. coli 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.
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.
