Part:BBa_K5103000
Cry8Da with PCG004 unique BsaI cut sites
Sequence and Features
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 1000INCOMPATIBLE 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 [1,2]
Usage and Biology
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).[1] 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, PCG004).
Figure 1. Sequence of Cry8Da gene with recognition sites. Image created using SnapGene.
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.
Previous research has looked at the genetic manipulation of Bt strains for increased insecticidal activity, and very few explore the expression of Cry8Da in bacterial strains, with most past studies exploring other related Cry proteins.[6] In particular, one particular study explores its expression in pET32a(+) within E. coli BL21(DE3)LysS.[7] They were able to induce protein expression with IPTG.[7] There has also been research exploring the expression of similar of Cry proteins and Cyt proteins within yeast.[8,9]. One study explored expressing Cry11A within S. cerevisiae.[8] Another study looked at the expression of Cyt2Aa1 in Pichia pastoris, using a synthetic version of the cytotoxic gene.[9]
Confirmation & Testing
An agarose gel electrophoresis was performed to confirm the successful ligation of Cry8Da in PCG004. This brings our team one step closer to producing a biopesticide of selective target of the Emerald Ash Borer (EAB) and closely related insects. See Part:BBa_K5103001 for more details.
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] 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
[2] 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
[3] 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
[4] 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
[5] Missouri iGEM. (2018). Missouri iGEM Project. https://2018.igem.org/Team:Missouri_Rolla
[6] George, Z. O. (2011). Expression and genetic manipulation of Bacillus thuringiensis toxins for improved toxicity and development of a protocol for in vivo selection of toxin variants with improved activity (Version 1). University of Sussex. https://hdl.handle.net/10779/uos.23316650.v1
[7] Thanh, L. T. M. (2012). Expression and purification of Cry8Da recombinant protein against coleopteran insects of bacillus thuringiensis in E. Coli. Vietnam Journal of Science and Technology, 50(3), 309–317. https://doi.org/10.15625/0866-708X/50/3/9500
[8] Quintana-Castro, R., Ramírez-Suero, M., Moreno, & Ramírez-Lepe, M. (2005). Expression of the cry11A gene of Bacillus thuringiensis ssp. israelensis in Saccharomyces cerevisiae. Canadian Journal of Microbiology, 51, 165-170. https://doi.org/10.1139/w04-126
[9] Gurkan, C., & Ellar, D. J. (2010). Expression of Bacillus thurigiensis Cyt2Aa1 toxin in Pichia pastoris using a synthetic gene construct. Biotechnology and Applied Biochemistry, 38(1), 25-33. https://doi.org/10.1042/BA20030017
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