Part:BBa_K3431034
oz21_B Toehold Switch for miR-21 Detection
Introduction
oz21_B toehold switch is a regulatory part for the downstream reporter gene. With this part, the protein expression can be controlled by the miR-21. The sequence of the toehold switch can be separated into the following 5 regions from its 5' end: TBS (trigger binding site), stem region, loop region with RBS (ribosome binding site), complimentary stem region with a start codon, and linker. Upon binding with miR-21, its hairpin structure can be opened up and the ribosomes can bind with its RBS (ribosome binding site), triggering the translation of the downstream reporter.
Design
The design of the toehold switch was mainly based on the previous research. 1 2 3 4 5 6 For the oz21_B toehold switch, we adopted the loop structure from Green et al., 2014 7, and the linker structure is from Green et al., 2016 8 . Using NUPACK analysis and Vienna binding models, we designed the sequence of the toehold switch. (See our model page: https://2020.igem.org/Team:CSMU_Taiwan/Model )
Characterization using invertase
The 2020 iGEM CSMU-Taiwan characterized the toehold switch with invertase (BBa_K3431000) reporter protein. The plasmid would be transcribed and translated with the protein synthesis kit at 37℃ for 2 hours. We would then add 5μl of 0.5M sucrose and measured the glucose concentration with Rightest TM GS550 glucose meter after 30 minutes. In our
experiments, the ON state refers to the conditions with miRNA triggers; while the OFF state means that there was no miRNA in the environment. We
calculated the ON/OFF ratio of the toehold switch, which is defined as “the glucose concentration of the ON state/ the glucose concentration of the OFF state”.
Results The ON/OFF ratio with miR-21 is 1.61, which suggested the regulatory function of the toehold switch. Thus, oz21_B toehold switch-regulated invertase can be controlled by the miR-21.
Reference
1. 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. https://doi.org/10.1016/j.cell.2014.10.002 2. Green, A. A., Kim, J., Ma, D., Silver, P. A., Collins, J. J., & Yin, P. (2017).Complex cellular logic computation using ribocomputing devices. Nature, 548(7665), 117–121. https://doi.org/10.1038/nature23271 3. Pardee, K., Green, A. A., Takahashi, M. K., Braff, D., Lambert, G., Lee, J.W., Ferrante, T., Ma, D., Donghia, N., Fan, M., Daringer, N. M., Bosch, I.,Dudley, D. M., O'Connor, D. H., Gehrke, L., & Collins, J. J. (2016). Rapid,Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell, 165(5), 1255–1266. https://doi.org/10.1016/j.cell.2016.04.059 4. Chappell, J., Westbrook, A., Verosloff, M., & Lucks, J. B. (2017). Computational design of small transcription activating RNAs for versatile and dynamic gene regulation. Nature communications, 8(1), 1051. https://doi.org/10.1038/s41467-017-01082-6 5. Sadat Mousavi, P., Smith, S. J., Chen, J. B., Karlikow, M., Tinafar, A., Robinson, C., Liu, W., Ma, D., Green, A. A., Kelley, S. O., & Pardee, K.(2020). A multiplexed, electrochemical interface for gene-circuit-based sensors. Nature chemistry, 12(1), 48–55. https://doi.org/10.1038/s41557-019-0366-y 6. Hong, F., Ma, D., Wu, K., Mina, L. A., Luiten, R. C., Liu, Y., Yan, H., &Green, A. A. (2020). Precise and Programmable Detection of Mutations Using Ultraspecific Riboregulators. Cell, 180(5), 1018–1032.e16. https://doi.org/10.1016/j.cell.2020.02.011 7. Green AA, Silver PA, Collins JJ, Yin P. Toehold switches: de-novo-designed regulators of gene expression. Cell 2014; 159(4): 925-39. 8. Pardee K, Green AA, Takahashi MK, et al. Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell 2016; 165(5): 1255-66.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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