Part:BBa_K4321007:Design

Cyt1Aa Cassette

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]

Design Notes

We designed our cassettes with flanking BsaI restriction sites to clone our cassettes downstream of a strong artificial promoter named Pgrac that was contained within our E.coli to Bacillus subtilis shuttle plasmid, pCG004. This cassette however, does not contain these restriction sites or the Pgrac feature so that future iGEM teams may clone this cassette into their desired plasmid under the control of their chosen promoter.

Unlike Cyt2Ba, the stability of the Cyt1Aa protein and colony formation in host and E.coli cells are reliant on the co-expression of a P20 helper protein. When trying to express Cyt1Aa, this factor should be considered. For our project we designed this cassette with flanking BsaI sites (included in cassette), for insertion into pCG004. However, desired restriction sites can be added to both ends of the cassette for desired plasmid insertion. Following the digestion with BsaI the last two bases will be lost to yield a 2206 bp fragment.

Source

The cassette was synthesized by IDT. Information on the origin of each part can be found on their respective part pages: P20 helpher protein - BBa_K2938004, Cyt1Aa - BBa_K2938003, RBS - BBa_B0034, mScarlet - BBa_K4321004, Terminator - BBa_K4321008.

References

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

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