Difference between revisions of "Part:BBa K3431001"

Revision as of 16:26, 23 October 2020

pp21_B Toehold Switch for miR-21 Detection

Description

This toehold switch has been designed to open up its hairpin loop structure upon binding with miRNA-21, 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-21, we optimised the region of loop with RBS and linker amino acids based on three articles: the original work on toehold switch (Green, A.A. et al., 2014), the adaptation of toehold switch to detect zika virus (Pardee, K. et al., 2016), and novel toehold switch design for detection of miRNA in mammalian cells (Wang, S. et al., 2019). We chose to test the 3 different loop structures and 2 different linker structures (Pardee, K. et al. and Wang, S. et al.) from the above-mentioned studies.

For this particular toehold switch (pp21_B), We incorporate the loop and linker sequence from work of Wang, S. et al. to test out whether optimisations specific to mammalian cells can have a better regulatory function for downstream protein expression in PURExpress in vitro protein synthesis system, on which our project of diagnosing oral cancer with toehold switch and glucose meter is based.

Model

NUPACK ANALYSIS

Experiment result

Reference

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. Wang, S., Emery, N. J., & Liu, A. P. (2019). A novel synthetic toehold switch for microRNA detection in mammalian cells. ACS synthetic biology, 8(5), 1079-1088.

Sequence and Features