Part:BBa_K2094001

Diaphorase (cytochrome b5 reductase)

Diaphorase is an enzyme that oxidizes NAD(P)H. With the right mediator such as Vitamin K, it can be used to generate electrity in a biobattery device.

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. Lastly, 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 comparison.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 358
  Illegal AgeI site found at 617
  
• 1000
  COMPATIBLE WITH RFC[1000]