Regulatory

Part:BBa_K3431036

Designed by: Jian-An Pan, Cheng-Yang Ma, Yi-Ching Chen, Shen-Lin Chen, Huan-Jui Chang   Group: iGEM20_CSMU_Taiwan   (2020-10-17)
Revision as of 07:56, 22 October 2020 by IrisRChen (Talk | contribs)


zz146_B Toehold Switch for miR-146 Detection

This toehold switch has been designed to open up its hairpin loop structure upon binding with miRNA-146, resulting in the translation of downstream reporter protein. The design of toehold switch can be separated into the following 5 regions from its 5' end: trigger binding sites, stem region, loop region with RBS, complimentary stem region with start codon, and linker amino acids. In our constructions of toehold switches for miRNA-146, we optimise the toehold switch structure by altering their loop region and linker sequence. We incorporate two designs of the loop region from two articles: the original work on toehold switch (Green, A.A. et al., 2014) and the adaptation of toehold switch to detect zika virus (Pardee, K. et al., 2016). Pardee, K. et al. have truncated the loop structure from 19 base pairs in the original work conducted by Green, A.A. et al. to 12 base pairs in order to reduce the leakage of output expression. Hence we hope to observe an increase in the output's dynamic range by implementing the loop sequence utilised by Pardee, K. et al.. As for our selection on the linker sequences, we choose to test out the linker sequence from Pardee, K. et al. and a random linker sequence which we generated in order to minimize the free energy of toehold switch mRNA secondary structure.

For this particular toehold switch (zz146_B), we incorporate the loop and linker structure from Pardee, K. et al..

NUPACK ANALYSIS
T--CSMU_Taiwan--zz146_B_NU.png

VIENNA RNA PACKAGE
T--CSMU_Taiwan--zz146_B_Ve.png

Link to our model page: https://2020.igem.org/Team:CSMU_Taiwan/Model

References: Green, A. A., Silver, P. A., Collins, J. J., & Yin, P. (2014). Toehold switches: de-novo-designed regulators of gene expression. Cell, 159(4), 925-939. Pardee, K., Green, A. A., Takahashi, M. K., Braff, D., Lambert, G., Lee, J. W., ... & Daringer, N. M. (2016). Rapid, low-cost detection of Zika virus using programmable biomolecular components. Cell, 165(5), 1255-1266.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 9
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 9
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 9
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 9
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 9
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
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