Coding

Part:BBa_K5103000:Design

Designed by: Nikoo Mansourian   Group: iGEM24_Guelph   (2024-09-17)


Cry8Da with PCG004 unique BsaI cut sites


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


Design Notes

BsaI is a type IIS digestion enzyme, which creates overhangs[1], so we must ensure that the sequences where the enzyme cuts on Cry8Da and PCG004 (refer to Part:BBa_K5103005) are complementary to each other. After designing and incorporating the cut sites with the Cry8Da[2] sequence, this fragment was ordered through IDT.

Source

The sequence of Cry8Da can be found in the NCBI GenBank under accession number KF148683.[3] The Cry8Da gene originates from the bacterium Bacillus thuringiensis (Bt), which is well known for producing Cry proteins.[4]

References

[1] New England Biolabs (2020). NEB.com. https://www.neb.com/en/products/r0535-bsai
[2] Gilbert, C., Howarth, M., Harwood, C. R., & Ellis, T. (2017). Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis. ACS synthetic biology, 6(6), 957–967. https://doi.org/10.1021/acssynbio.6b00292
[3] Xie, B., & Liu, Y. (2013). Cry8Da [Bacillus thuringiensis]. NCBI. https://www.ncbi.nlm.nih.gov/protein/543174970
[4] Shu, C., Yan, G., & Soberón, M. (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