Part:BBa_K4321007

Cyt1Aa Cassette

Since its discovery in 1901, Bacillus thuringiensis (Bt) has been identified as encoding over 800 insecticidal crystal proteins (ICP). These ICPs include delta endotoxins of the crystal (Cry) and cytolytic (Cyt) family.

Cyt proteins can be split into three subfamilies (Cyt1, Cyt2, and Cyt3) and all have the same mode of action. They are produced as protoxins and ultimately activated by species-specific proteolytic cleavage in the midgut of insect targets. Activated Cyt1Aa binds to membrane receptors to signal increased cell permeability. This promotes pore formation in the lining of the midgut and ultimately kills the target insect.

Unlike Cry proteins, Cyt proteins are toxic only to Diptera species and can potentially suppress the resistance to Cry proteins. These proteins can be used in their native structure or be modified to target insect species outside of the Diptera suborder.

Usage and Biology

Upon ingestion of the Cyt1Aa protoxin, Cyt1Aa is solubilized in the gut of Dipteran insects. Once solubilized the protoxin is proteolytically cleaved into the active 22-25kDa protein. The active Cyt1Aa toxin inserts itself into the apical microvilli membrane of epithelial cells in the midgut and causes the death of the insect. This is hypothesized to be done through one of two mechanisms. The first includes Cyt1Aa binding to the epithelial membrane and promoting the formation of cationic channels that result in an influx of water into the cell, swelling, and ultimately lysis. The Second mechanism involves the aggregation of Cyt1Aa on the lipid bilayer which leads to membrane disassembly and cell death.

Outcome of Toxicity Assay

A toxicity assay was conducted with the Cyt1Aa-pCG004 transformed B.subtilis cells. Bacillus subtilis expression plasmids tend to have leaky expression in E.coli cells. This often results in the unexpected and oftentimes high expression of proteins. In this case, transformed DH5alpha E.coli cells fluoresced red due to mScarlet's absorption maxima being 569 nanometers, which falls in the white light spectrum. Due to the leaky expression our cassettes in E.coli, Cyt1Aa-pCG004 transformed cells were also tested. Measurements were taken in 2 minute intervals until the death of our Diptera insect species (Drosophila melanogaster) was observed.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 264
  Illegal PstI site found at 1006
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 264
  Illegal PstI site found at 1006
  Illegal NotI site found at 1888
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 264
  Illegal BglII site found at 1304
  Illegal BamHI site found at 683
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 264
  Illegal PstI site found at 1006
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 264
  Illegal PstI site found at 1006
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

PCG004 (plasmid #87377). Addgene. (n.d.). Retrieved October 8, 2022, from https://www.addgene.org/87377/

Soberón, M., López-Díaz, J. A., & Bravo, A. (2013). Cyt toxins produced by bacillus thuringiensis: A protein fold conserved in several pathogenic microorganisms. Peptides, 41, 87–93. https://doi.org/10.1016/j.peptides.2012.05.023

Torres-Quintero, M.-C., Gómez, I., Pacheco, S., Sánchez, J., Flores, H., Osuna, J., Mendoza, G., Soberón, M., & Bravo, A. (2018). Engineering bacillus thuringiensis cyt1aa toxin specificity from dipteran to lepidopteran toxicity. Scientific Reports, 8(1). https://doi.org/10.1038/s41598-018-22740-9

Tran, D. T., Phan, T. T., Doan, T. T., Tran, T. L., Schumann, W., & Nguyen, H. D. (2020). Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in bacillus subtilis. Biotechnology Reports, 28. https://doi.org/10.1016/j.btre.2020.e00540

Valtierra-de-Luis, D., Villanueva, M., Berry, C., & Caballero, P. (2020). Potential for bacillus thuringiensis and other bacterial toxins as biological control agents to combat dipteran pests of medical and agronomic importance. Toxins, 12(12), 773. https://doi.org/10.3390/toxins12120773