Part:BBa_K4882004

Pmcl1-SuperNova-SV40 NLS-TtrpC

This is a complete suicide switch cassette consisting of a hemolymph inducible promoter Pmc1, a red fluorescent protein SuperNova fused with an SV40 nuclear localization signal (NLS), and a trpC terminator.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1673
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 3486
  Illegal XhoI site found at 853
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1229
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1179
  Illegal BsaI.rc site found at 2923
  Illegal BsaI.rc site found at 3214
  Illegal SapI site found at 1130
  

Usage and Biology

Metarhizium anisopliae is an entomopathogenic fungus widely used as a biopesticide. To improve the safety of M. anisopliae as a fungal biopesticide, we decided to introduce a suicide switch into the fungus. SuperNova (BBa_K4882008) is a red fluorescent protein that produces reactive oxygen species (ROS) in the presence of green-yellow light (~579 nm). The SV40 NLS (BBa_K2549054) should guide the protein to be transported into the cell nucleus, and let ROS attack the most vulnerable genomic DNA (Paardekooper et al., 2019). The SuperNova-SV40 NLS (BBa_K4882003) is linked downstream of a hemolymph inducible promoter Pmcl1 (BBa_K2040100). When the fungi invade the insect body, Pmcl1 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. A trpC terminator was also included to form the complete expression cassette.

Characterization

2023 Hangzhou-SDG Team characterized this part as a suicide switch We tested the suicide switches based on the amounts of spore formation and the spore germination rates. Engineered strains were inoculated to the larvae of the greater wax moth (Galleria mellonella). The Petri dishes containing the pest 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 1. A. The amount of spore formation by each strain on G. mellonella corpses; B. The spore germination rate of each strain in liquid PDB media after 10h. *: p < 0.01; **: p < 0.001.

The results showed that the strain containing Pmcl1-SuperNova-SV40 NLS-trpC terminator formed significantly fewer spores than the WT strain (p < 0.01).

After being collected, the spores from each strain were resuspended in liquid PDB media at the concentration of 5 × 10^5 spores/mL. The suspensions were incubated at 30 °C, 180 rpm for 10h to facilitate spore germination.

The germination rate results indicated that Pmcl1-SuperNova-SV40 NLS-trpC terminator could not influence spore viability after spore formation.

References

Paardekooper LM, van Vroonhoven E, Ter Beest M, van den Bogaart G. Radical Stress Is More Cytotoxic in the Nucleus than in Other Organelles. Int J Mol Sci. 2019 Aug 25;20(17):4147. doi: 10.3390/ijms20174147. PMID: 31450682; PMCID: PMC6747261.