Part:BBa_K5066007
Cyt2Ba-p20-Xpp81Aa1
Description
Insecticide resistance is becoming increasingly problematic and prominent in many Southeast Asian countries, areas where dengue fever is prevalent. The combination of Bt toxins and scorpion toxins is a promising strategy providing a potential layer of protection against insecticide resistance. The ribosome binding site(RBS) is a sequence of nucleotides before the start codon of an mRNA transcript and functions to recruit ribosomes for the translation of proteins. The His-tag, 6X His, is a commonly used purification tag that contains 6 consecutive histidine residues. The His-tag can be stained with His-tag antibodies after translation, labelling the target recombinant protein. It is typically placed on either the N or C terminus of a protein. The strain also has its stop codons removed in order to increase the production of toxins. P20 is a chaperone that aids in the production of Bt toxins.
Fig 1. Plasmid construct design in pET-28a
Use in Biology
Cyt2Ba is a toxin that binds to membrane receptors and increases membrane permeability. In Aedes mosquitoes, the toxin binds to the midgut of the mosquito larvae. Due to its crystalline structure, the toxin alters the cell membrane’s permeability which is crucial for cellular transport and activities, ultimately leading to the death of the larvae as a result of malnutrition and damage to the cell membrane.[1][2][3] Xpp81Aa1 is one of the Bacillus thuringiensis toxins, or Bt toxins, that derive from Bt bacteria and are commonly used as insecticides as they can target specific insects without causing harm to other species. There are a wide variety of strains derived from a selection of Bt bacteria and each has similar effects but targets different species of insects. Previous studies have demonstrated its benefit in assisting the mortality rate of mosquito larvae is its symbiotic relationship with Bt toxins (such as Cry2Aa and Cry4Aa). Chosen for its coactive properties, Xpp81Aa1 heightens the effectiveness of the other toxins.[2]
Table 2. The mortality rate of Aedes albopictus larvae exposed to various combinations of biolarvicidal toxins.
Note: Both Cyt2Ba and Xpp81Aa1 are basic parts that iGEM24_GEMS-Taiwan has characterized. Their codes are the following
Cyt2Ba: BBa_K5066000
Xpp81Aa1: BBa_K5066001
On the other hand, p20 was derived from iGEM19_BGU_Israel: BBa_K2938004
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 485
Illegal EcoRI site found at 879
Illegal EcoRI site found at 1192 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 485
Illegal EcoRI site found at 879
Illegal EcoRI site found at 1192 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 485
Illegal EcoRI site found at 879
Illegal EcoRI site found at 1192
Illegal BglII site found at 1640 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 485
Illegal EcoRI site found at 879
Illegal EcoRI site found at 1192 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 485
Illegal EcoRI site found at 879
Illegal EcoRI site found at 1192 - 1000COMPATIBLE WITH RFC[1000]
Reference
[1] Bravo, A., Likitvivatanavong, S., Gill, S. S., & Soberón, M. (2011). Bacillus thuringiensis: A story of a successful bioinsecticide. Insect Biochemistry and Molecular Biology, 41(7), 423–431. https://doi.org/10.1016/j.ibmb.2011.02.006
[2] Wu, J., Wei, L., He, J., Fu, K., Li, X., Jia, L., Wang, R., & Zhang, W. (2021). Characterization of a novel Bacillus thuringiensis toxin active against Aedes aegypti larvae. Acta Tropica, 223, 106088. https://doi.org/10.1016/j.actatropica.2021.106088
[3] Gu, J.-B., Dong, Y.-Q., Peng, H.-J., & Chen, X.-G. (2010). A Recombinant AeDNA Containing the Insect-Specific Toxin, BmK IT1, Displayed an Increasing Pathogenicity on Aedes albopictus. American Journal of Tropical Medicine and Hygiene, 83(3), 614–623. https://doi.org/10.4269/ajtmh.2010.10-0074
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