Part:BBa_K3431013

or146_A Toehold Switch for miR-146 Detection

Description

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 (or146_A), we incorporate the loop structure from Green, A.A. et al. and the random linker structure generated by our team.

Model

NUPACK ANALYSIS

VIENNA RNA PACKAGE

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

Experiment result

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]