Composite

Part:BBa_K5103001:Design

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


PCG004 with Cry8Da


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
    Illegal EcoRI site found at 8948
    Illegal EcoRI site found at 9027
    Illegal EcoRI site found at 10439
    Illegal EcoRI site found at 11102
    Illegal XbaI site found at 11533
    Illegal PstI site found at 8079
    Illegal PstI site found at 9520
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
    Illegal EcoRI site found at 8948
    Illegal EcoRI site found at 9027
    Illegal EcoRI site found at 10439
    Illegal EcoRI site found at 11102
    Illegal NheI site found at 6234
    Illegal PstI site found at 8079
    Illegal PstI site found at 9520
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
    Illegal EcoRI site found at 8948
    Illegal EcoRI site found at 9027
    Illegal EcoRI site found at 10439
    Illegal EcoRI site found at 11102
    Illegal BglII site found at 66
    Illegal BglII site found at 3147
    Illegal XhoI site found at 3151
    Illegal XhoI site found at 11410
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
    Illegal EcoRI site found at 8948
    Illegal EcoRI site found at 9027
    Illegal EcoRI site found at 10439
    Illegal EcoRI site found at 11102
    Illegal XbaI site found at 11533
    Illegal PstI site found at 8079
    Illegal PstI site found at 9520
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
    Illegal EcoRI site found at 8948
    Illegal EcoRI site found at 9027
    Illegal EcoRI site found at 10439
    Illegal EcoRI site found at 11102
    Illegal XbaI site found at 11533
    Illegal PstI site found at 8079
    Illegal PstI site found at 9520
    Illegal AgeI site found at 9294
    Illegal AgeI site found at 10176
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 11524
    Illegal BsaI.rc site found at 7949
    Illegal BsaI.rc site found at 7962
    Illegal BsaI.rc site found at 11537
    Illegal SapI.rc site found at 1219
    Illegal SapI.rc site found at 5124


Design Notes

The GFP sequence of PCG004[1] is cut out using BsaI and replaced by Cry8Da also digested with BsaI. See Part:BBa_K5103000 and Part:BBa_K5103005. This allows us to induce the expression of Cry8Da using IPTG to induce the expression when needed.


Figure 1. PCG-CRY plasmid components were developed using SnapGene.


The components in this plasmid are as follows:

  • Cry8Da gene: Insecticidal protein gene that has been modified to be transformed into PCG004. See BBa_K5103000.
  • Ampicillin resistance: Selection marker for transformed E. coli.
  • Lac operon: IPTG-inducible promoter, controls Cry8Da expression in E. coli.

Source

The sequences for this part are sourced from NCBI[2] and Addgene[1].

References

[1] 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
[2] Xie, B., & Liu, Y. (2013). Cry8Da [Bacillus thuringiensis]. https://www.ncbi.nlm.nih.gov/protein/543174970