Coding

Part:BBa_K5103000

Designed by: Nikoo Mansourian   Group: iGEM24_Guelph   (2024-09-17)
Revision as of 16:10, 30 September 2024 by Aadelino (Talk | contribs) (→‎Profile)


Cry8Da with PCG004 unique BsaI cut sites

Cry8Da for PCG004

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 993
    Illegal EcoRI site found at 1072
    Illegal EcoRI site found at 2484
    Illegal EcoRI site found at 3147
    Illegal PstI site found at 124
    Illegal PstI site found at 1565
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 993
    Illegal EcoRI site found at 1072
    Illegal EcoRI site found at 2484
    Illegal EcoRI site found at 3147
    Illegal PstI site found at 124
    Illegal PstI site found at 1565
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 993
    Illegal EcoRI site found at 1072
    Illegal EcoRI site found at 2484
    Illegal EcoRI site found at 3147
    Illegal XhoI site found at 3455
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 993
    Illegal EcoRI site found at 1072
    Illegal EcoRI site found at 2484
    Illegal EcoRI site found at 3147
    Illegal PstI site found at 124
    Illegal PstI site found at 1565
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 993
    Illegal EcoRI site found at 1072
    Illegal EcoRI site found at 2484
    Illegal EcoRI site found at 3147
    Illegal PstI site found at 124
    Illegal PstI site found at 1565
    Illegal AgeI site found at 1339
    Illegal AgeI site found at 2221
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 3569
    Illegal BsaI.rc site found at 7


Profile

Name: Cry8Da
Base Pairs: 3580 bp
Origin: Bacillus Thuringensis
Properties: Cytoxin which induces proliferation in insect guts and causes death

Usage and Biology

Figure 1. Sequence of Cry8Da gene with recognition sites.

This part contains the Cry8Da gene, which encodes a protein known for its insecticidal properties, specifically targeting members of the Coleoptera order, including the emerald ash borer (EAB). The Cry8Da sequence is flanked by BsaI restriction sites, facilitating its integration into the pGC004 plasmid backbone. Additionally, the sequence includes HindIII, XhoI, and BsaI cut sites at the 3' end, as well as BsaI and NdeI cut sites at the 5' end. These modifications enable seamless integration of Cry8Da into all the plasmids utilized in our project (pYES2, pET28a, PCGOO4). Research has shown that Cry8Da effectively kills insects in the Coleoptera order.[1] The protein works by binding to receptors in the insect's midgut, leading to pore formation and eventual cell death.[1,2] The toxic domain of the Cry8Da protein is thought to be its 54 kDa active fragment, which is generated through proteolytic cleavage.[3,4] Cry8Da is activated through proteolytic cleavage, and the resulting fragments are essential for the toxin’s insecticidal action, with different domains responsible for binding to receptors (C-terminal) and pore formation (N-terminal) in the gut cell of the beetle.[4] Cry8Da gets cleaved into 3 fragments: 64kDa, 54kDa and 8kDa. The 54kDa and the 8kDa fragments are generated by intramolecular cleavage at the loop between the α3 and α4 helices of domain I. The 54kDa fragment is the primary binding fragment responsible for interacting with receptors in the insect's gut such as ß-glucosidase. This interaction is what causes the mechanism of action leading to pore formation in the cell membrane and insect death.[3,4] In 2018, iGEM Missouri attempted to use Cry8Da, a cytotoxin with specificity against the EAB by binding to the receptors on the gut epithelial cells of the EAB thus causing cell lysis and death, to protect Ash trees through genetic engineering Arabidopsis thaliana (A. thaliana), a model organism.[5] However, the Missouri team was unsuccessful in combining their parts and were unable to develop EAB-resistant plants. As such, no other iGEM team has successfully or otherwise used the Cry8Da protein in their project. Figure 2. 0.8% Agarose gel electrophoresis of PCG-CRY plasmid digested with BsaI compared to the undigested PCG-CRY plasmid. Digestion shows successful ligation of the Cry8Da gene in pCG004 plasmid backbone. Results showed a band at ~8000bp corresponding with PCG004 and ~3400 bp corresponding with Cry8Da.

References

[1] Missouri iGEM. (2018). Missouri iGEM Project. https://2018.igem.org/Team:Missouri_Rolla

[2] Shu, C., Yan, G., Huang, S., Geng, Y., Soberón, M., Bravo, A., Geng, L., & Zhang, J. (2020). Characterization of two novel Bacillus thuringiensis Cry8 toxins reveals differential specificity of protoxins or activated toxins against Chrysomeloidea coleopteran superfamily. Toxins, 12(10), 642. https://doi.org/10.3390/toxins12100642

[3] Bravo, A., Gill, S. S., & Soberón, M. (2007). Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon, 49(4), 423-435. https://doi.org/10.1016/j.toxicon.2006.11.022

[4] Yamaguchi, T., Bando, H., & Asano, S. (2013). Identification of a Bacillus thuringiensis Cry8Da toxin-binding glucosidase from the adult Japanese beetle, Popillia japonica. Journal of Invertebrate Pathology, 113(2), 123-128. https://doi.org/10.1016/j.jip.2013.03.006

[5] Yamaguchi, T., Sahara, K., Bando, H., & Asano, S. (2010). Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle. Journal of Invertebrate Pathology, 105(3), 243-247. https://doi.org/10.1016/j.jip.2010.07.002

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