Difference between revisions of "Part:BBa K4321009"
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===Outcomes of Toxicity Assay=== | ===Outcomes of Toxicity Assay=== | ||
| − | https://static.igem.org/mediawiki/parts | + | https://static.igem.org/mediawiki/parts/4/4d/Outcomes_of_Toxicity_assay.png |
| − | A toxicity assay was conducted with the | + | |
| + | A toxicity assay was conducted with the Cyt2Ba-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 did not fluoresce blue due to the BFP's absorption maxima being 380 nanometers, which falls outside the white light spectrum. Due to the leaky expression our cassettes in E.coli, Cyt2Ba-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=== | ===Sequence and Features=== | ||
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==References== | ==References== | ||
Cohen, S., Dym, O., Albeck, S., Ben-Dov, E., Cahan, R., Firer, M., & Zaritsky, A. (2008). High-resolution crystal structure of activated Cyt2Ba monomer from Bacillus thuringiensis subsp. israelensis. Journal of molecular biology, 380(5), 820–827. https://doi.org/10.1016/j.jmb.2008.05.010 | Cohen, S., Dym, O., Albeck, S., Ben-Dov, E., Cahan, R., Firer, M., & Zaritsky, A. (2008). High-resolution crystal structure of activated Cyt2Ba monomer from Bacillus thuringiensis subsp. israelensis. Journal of molecular biology, 380(5), 820–827. https://doi.org/10.1016/j.jmb.2008.05.010 | ||
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| + | 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 | 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 | ||
| + | |||
| + | 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 | 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 | ||
Wang, FF., Qu, SX., Lin, JS. et al. Identification of Cyt2Ba from a New Strain of Bacillus thuringiensis and Its Toxicity in Bradysia difformis. Curr Microbiol 77, 2859–2866 (2020). https://doi.org/10.1007/s00284-020-02018-y | Wang, FF., Qu, SX., Lin, JS. et al. Identification of Cyt2Ba from a New Strain of Bacillus thuringiensis and Its Toxicity in Bradysia difformis. Curr Microbiol 77, 2859–2866 (2020). https://doi.org/10.1007/s00284-020-02018-y | ||
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Latest revision as of 04:04, 12 October 2022
Cyt2Ba 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 Cyt2Ba 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 Cyt2Ba protoxin, Cyt2Ba is solubilized in the gut of Dipteran insects. Once solubilized the protoxin is proteolytically cleaved into the active 30 kDa protein. The active Cyt2Ba 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 Cyt2Ba 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 Cyt2Ba on the lipid bilayer which leads to membrane disassembly and cell death.
Outcomes of Toxicity Assay
A toxicity assay was conducted with the Cyt2Ba-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 did not fluoresce blue due to the BFP's absorption maxima being 380 nanometers, which falls outside the white light spectrum. Due to the leaky expression our cassettes in E.coli, Cyt2Ba-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
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References
Cohen, S., Dym, O., Albeck, S., Ben-Dov, E., Cahan, R., Firer, M., & Zaritsky, A. (2008). High-resolution crystal structure of activated Cyt2Ba monomer from Bacillus thuringiensis subsp. israelensis. Journal of molecular biology, 380(5), 820–827. https://doi.org/10.1016/j.jmb.2008.05.010
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
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
Wang, FF., Qu, SX., Lin, JS. et al. Identification of Cyt2Ba from a New Strain of Bacillus thuringiensis and Its Toxicity in Bradysia difformis. Curr Microbiol 77, 2859–2866 (2020). https://doi.org/10.1007/s00284-020-02018-y