Composite

Part:BBa_K3059638:Design

Designed by: Julia Urban   Group: iGEM19_William_and_Mary   (2019-10-20)


pBlind Curli Operon, Blue-Light Inducible


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2730
    Illegal PstI site found at 1829
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2730
    Illegal NheI site found at 51
    Illegal NheI site found at 74
    Illegal PstI site found at 1829
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2730
    Illegal XhoI site found at 607
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2730
    Illegal PstI site found at 1829
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2730
    Illegal PstI site found at 1829
    Illegal NgoMIV site found at 177
    Illegal AgeI site found at 3027
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This circuit incorporates the pBlind-EL222 system, which is sensitive to blue light. Constitutively expressed EL222 can activate pBlind under blue light, resulting in increased expression of the downstream curli operon. To assemble this circuit, we utilized 3G DNA assembly, which required individual 3G-compatible DNA parts (BsaI cutsites and proper sticky ends) as well as transcriptional units flanked by the proper Universal Nucleotide Sequences (UNS). To construct the transcriptional unit containing the synthetic curli circuit, we assembled csgBAC and csgEFG (which are divergent in E. coli) together, linking them with an RBS before csgE. This assembly required the creation of new sticky end (sticky end alpha, see parts pages BBa_K3059626, BBa_K3059627, and BBa_K3059612) in addition to William and Mary 2018 iGEM's sticky ends A-E.

Source

Our synthetic curli operon is composed of csgBAC, an RBS preceding csgE, and csgEFG. See BBa_K3059619, BBa_K305920, and BBa_K3059621, respectively for information the sources of these parts. Additional promoters, RBS, and terminators used in this circuit were chosen from the 3G DNA part library created by William and Mary 2018 iGEM.

References

Jayaraman, P., Devarajan, K., Chua, T. K., Zhang, H., Gunawan, E., & Poh, C. L. (2016). Blue light-mediated transcriptional activation and repression of gene expression in bacteria. Nucleic acids research, 44(14), 6994–7005. doi:10.1093/nar/gkw548