DNA

Part:BBa_K5066002:Design

Designed by: Abner Tseng, Megan Chiao, and Pin-Chen Ting   Group: iGEM24_GEMS-Taiwan   (2024-09-14)


BmK IT1


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We obtained the gene fragment from NCBI, conducted codon optimization before PCR amplification and inserting the sequence into the bacterial vector pET-28a, which includes a T7 promoter, ribosomal binding site, and a Lac operator. The pET series of expression plasmid is commonly used for recombinant protein production in E. coli, this is due to its T7 promoter and its adjacent Lac operator which helps suppress uninduced expression. The T7 promoter is a sequence of base pairs before the gene that acts as an indicator of where the RNA polymerase binds to begin the process of transcription. The ribosome binding site, represented as RBS on the vector, is a sequence of base pairs upstream of the start codon, allowing ribosome binding to the mRNA. The lac operator is an inhibitor which restricts the transcription of the gene, but the effects can be reversed by the binding of an inducer, in our project we used IPTG, which mimics an allolactose. The IPTG binds to the lac operator which induces the synthesis of protein

Source

The strain is extracted from the scorpion Buthus martensii Karsch obtained from NCBI

References

[1] 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

[2] Deng, S.-Q., Deng, M.-Z., Chen, J.-T., Zheng, L.-L., & Peng, H.-J. (2017). [Larvicidal activity of recombinant Escherichia coli expressing scorpion neurotoxin AaIT or B.t.i toxin Cyt2Ba against mosquito larvae and formulations for enhancing the effects]. Nan Fang Yi Ke Da Xue Xue Bao = Journal of Southern Medical University, 37(6). https://doi.org/10.3969/j.issn.1673-4254.2017.06.06

[3] Bougis, P. E., Kopeyan, C., Li, H. M., Luo, M. J., Xiong, Y. M., Barton, K. A., & Eitan, M. (1999, January 6). The cdna sequence of an excitatory insect selective neurotoxin from the scorpion buthus martensi karsch. Toxicon. https://www.sciencedirect.com/science/article/abs/pii/S0041010198001767