Part:BBa_K2588018

Alternative oxidase 1 from Nelumbo nucifera

NnAOX1 is the alternative oxidase 1 from Nelumbo nucifera with the function to salvage electrons from the respiratory chain for heat generation. In our experiments however, we failed to yield a significant heat production by usage of AOX1.

Usage and Biology

NnAOX1 is a very important part of the temperature regulation system in lotus, which is well-known for its precision at temperatures between 30 °C and 35 °C. Furthermore, AOX has been found to be an important ROS scavenger.¹ AOX1 can be utilized for the generation of heat, because it can salvage electrons from the respiratory chain. In this project it has been used in combination with oHybB as a composite part (BBa_K2588026), to increase the temperature in the mosquito trap if the surrounding temperature is low to attract mosquitoes.

Characterization

For the characterization of this part, a composite part with the arabinose-inducible pBAD-promotor was created (BBa_K2588027). Three 100 mL cultures of DH5α with pSB1C3-BBa_K206000-BBa_B0034-NnAOX1a were inoculated with an OD₆₀₀ = 0.1, incubated at 21 °C (room temperature) and induced by addition of 10 µM arabinose when an OD₆₀₀ = 0.2 was reached. Additionally, an untransformed DH5α control without the plasmid was cultured as a negative control. The experiment was performed at room temperature and the variation of temperature was measured in an interval of 1 h for 10 hours. Unexpectedly, there was no difference between the negative control and the induced AOX1 culture (Fig. 1). We confirmed our results in five different experiments, and all failed to show heat production induced by AOX1 expression.

Fig. 1:The temperature of DH5α with arabinose-induced AOX1 expression does not differ significantly from untransformed DH5α. p=0.913.

Disussion

Unfortunately, even after various experiments, we were not able to confirm that AOX1 is capable of heat production contradictory to results published by iGEM Team Georgia Tech 2010.

References

1. Yin Li, Y., Zhang, Y., Wei, S., Liu, L. & Chen, Y. Recovery of antioxidant gene expression in sacred lotus (Nelumbo nucifera Gaertn.) embryonic axes enhances tolerance to extreme high temperature. AFRICAN J. Biotechnol. 11, 12011–12019 (2012).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 84
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 221
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]