Regulatory

Part:BBa_K3431006

Designed by: Jian-An Pan, Cheng-Yang Ma, Yi-Ching Chen, Shen-Lin Chen, Huan-Jui Chang   Group: iGEM20_CSMU_Taiwan   (2020-07-22)


oz21_A Toehold Switch for miR-21 Detection

Introduction

oz21_A 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_A 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 )

Figure 1. NUPACK analysis result
Figure 2. ViennaRNA Package result
















Characrterization 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”.

Figure 3. The glucose productions of the oz21_A toehold switch-regulated invertase in different states. The blue bar refers to the OFF state (not added with miRNA). The green bar refers to the ON state (added with miR-21 trigger). The yellow bar refers to the state with non-related RNAs (added with miR-191). The pink bar refers to the state with non-related RNAs (added with miR-223).

Results The ON/OFF ratio with miR-21 is 12.3, which suggested the regulatory function of the toehold switch. Thus, oz21_A toehold switch-regulated invertase can be controlled by the miR-21.

References

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.

Information contributed by City of London UK (2021)

ToeholdTools.png

This toehold switch was characterized in silico using the ToeholdTools project that our team developed. See https://github.com/lkn849/thtools for more information.

Metadata:

  • Group: City of London UK 2021
  • Author: Lucas Ng
  • Summary: Used our software ToeholdTools to investigate the target miRNA specificity and activation of this part.

Raw data:

This contribution was autogenerated by the script contrib.py, available at https://github.com/lkn849/thtools/tree/master/registry.

This switch was designed to detect the miRNA hsa-miR-21-5p at a temperature of 37°C. We tested it against every mature Homo sapiens miRNA in miRBase and our analysis shows that at this temperature it is best used to detect hsa-miR-21-5p.

With hsa-miR-21-5p at 37°C, the switch has a specificity of 1 ± 100 % and an activation of 8 ± 5 %. These values represent 95% confidence limits (z=1.96).

The temperature–activation–specificity relationship is shown here. CRT is an acronym for CelsiusRangeTest, the class in our Python library responsible for the following graph:

BBa K3431006 graph.png

Error bars represent the standard deviation. The line of best fit was calculated using a univariate cubic spline weighted inverse to each point's standard error.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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