Part:BBa_K4882008

SuperNova

SuperNova is a red fluorescent protein that produces reactive oxygen species (ROS) in the presence of green-yellow light (~579 nm) (Takemoto et al., 2013). Supernova is the monomeric version of KillerRed (BBa_K1184000) and should produce three times as much ROS as KillerRed can (Onukwufor et al., 2019).

Figure 1. The 3D structure of SuperNova (PDB ID: 3WCK; Takemoto et al., 2013).

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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 151
  Illegal BsaI.rc site found at 442
  

Usage and Biology

Metarhizium anisopliae is an entomopathogenic fungus widely used as a biopesticide. To improve its safety, SuperNova is used as a part of a suicide switch for the engineered M. anisopliae. SuperNova is linked downstream of a hemolymph inducible promoter Pmcl1 (short, BBa_K4882000). When the fungi invade the insect body, Pmcl1(short) turns on, and they cumulate SuperNova protein. When the fungi grow out of the insect body and try to spread spores, the tissues are under sunlight and killed by ROS.

Characterization

2023 Hangzhou-SDG Team characterized this part as part of a suicide switch

SuperNova can be a part of the suicide switch for M. anisopliae when being connected after the hemolymph inducible promoter Pmcl1/Pmcl1(short).

Larvae of Galleria mellonella were killed by engineered M. anisopliae. The corpses were placed under sunshine for days until spores formed outside the insect body. Spores were collected by vertexing and the concentrations of spore suspensions were checked under a microscope.

Figure 2. The amount of spore formation by M. anisopliae containing SuperNova on G. mellonella corpses. **: p < 0.01; ***: p < 0.001; ****: p < 0.0001.

Results showed that suicide switches consisting of Supernova led to significantly fewer spores than WT did.

References

Onukwufor, J. O.; Trewin, A. J.; Baran, T. M.; Almast, A.; Foster, T. H.; Wojtovich, A. P. Quantification of Reactive Oxygen Species Production by the Red Fluorescent Proteins KillerRed, Supernova, and mCherry. Free Radical Biology and Medicine 2020, 147, 1–7. DOI:10.1016/j.freeradbiomed.2019.12.008.

Takemoto, K., Matsuda, T., Sakai, N. et al. SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation. Sci Rep 3, 2629 (2013). https://doi.org/10.1038/srep02629