Coding

Part:BBa_K2200007:Design

Designed by: Mixiao Gui   Group: iGEM17_Shenzhen_SFLS   (2017-09-29)
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Gal4 transcription factor is a positive regulator.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 710
    Illegal XhoI site found at 218
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 886
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 137


Design Notes

Our modeling results showed that our sgRNA sequence has high cleavage efficiency on the mutated BRAF gene, as well as a high risk of off-target effect. To avoid the off-target effect on normal cells, we tried hard to develop an regulatory system for the CRISPR/Cas9 system. At first, we thought about simply promote the system by the mutant hTERT but found it is a weak promoter. We then considered enhancing the function of the promoter. We focused on the NF-KB p65 protein. However, we should change our direction since we found an article with complete results and their design is exactly the same as ours. So we have to look for other directions (that is, our present regulate system,for more information please enter http://2017.igem.org/Team:Shenzhen_SFLS/Design).

We thought the Gal4-p65 could still be an improvement for BBa_K1470002(https://parts.igem.org/Part:BBa_K1470002) although we didn’t utilize it in our final project.


Source

It comes from Saccharomyces cerevisiae.

References

[1] Huang X, Zhuang C, Zhuang C, et al. An enhanced hTERT promoter-driven CRISPR/Cas9 system selectively inhibits the progression of bladder cancer cells. Molecular Biosystems, 2017, 13(9): 1713-1721.