Difference between revisions of "Part:BBa K3431001"

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<partinfo>BBa_K3431001 short</partinfo>
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<partinfo>BBa_K3431007 short</partinfo>
  
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===Description===
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=== Introduction ===
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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.
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pp21_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.
  
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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.
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===Design===
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The design of the toehold switch was mainly based on the previous research<sup>[1][2][3][4][5][6]</sup>. For the pp21_B toehold switch, we adopted the loop and the linker structure from Wang et al., 2019<sup>[7]</sup>. 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 )
  
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===Model===
 
 
<html>
 
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NUPACK ANALYSIS <br>
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<figure style="mirgin-right: 1em; float:left; width:40%; border:1px solid black">
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<div style="width=100%; display:flex; align-items: center; justify-content: center;">
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<img src="https://static.igem.org/mediawiki/parts/3/31/T--CSMU_Taiwan--pp21_B_NU.png" style="display: block;margin-left: auto;margin-right: auto; width: 70%">
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<img src="https://static.igem.org/mediawiki/parts/3/31/T--CSMU_Taiwan--pp21_B_NU.png" style="width:50%;">
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<figcaption style="text-align: center;">
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Figure 1. NUPACK analysis result
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</figcaption>
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</figure>
 
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<br>
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<figure style="mirgin-right: 1em; float:left; width:40%; border:1px solid black">
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<img src="https://static.igem.org/mediawiki/parts/b/b9/T--CSMU_Taiwan--pp21_B_VE.png" style="display: block;margin-left: auto;margin-right: auto; width: 100%">
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<figcaption style="text-align: center;">
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Figure. 2. ViennaRNA Package result
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</figcaption>
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</figure>
 
</html>
 
</html>
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VIENNA RNA PACKAGE<br>
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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
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<html>
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===Characterization using invertase===
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<div style="width=100%; display:flex; align-items: center; justify-content: center;">
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<img src="https://static.igem.org/mediawiki/parts/b/b9/T--CSMU_Taiwan--pp21_B_VE.png" style="width:50%;">
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</div>
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<br>
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</html>
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Link to our model page: https://2020.igem.org/Team:CSMU_Taiwan/Model
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===Experiment result===
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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 RightestTM 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”.
 
<html>
 
<html>
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<br>
 
<div style="width=100%; display:flex; align-items: center; justify-content: center">
 
<div style="width=100%; display:flex; align-items: center; justify-content: center">
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<img src="https://static.igem.org/mediawiki/parts/e/e5/T--CSMU_Taiwan--pp21_B_%28BBa_K3431001%29.png" style="width:50%">
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<img src="https://static.igem.org/mediawiki/parts/e/e5/T--CSMU_Taiwan--pp21_B_%28BBa_K3431001%29.png" style="width:40%">
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<figcaption style="text-align: center;">
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</figcaption>
 
</div>
 
</div>
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Figure. 3. The glucose productions of the pp21_B 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).
 
<br>
 
<br>
 
</html>
 
</html>
  
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===Reference===
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<b>Results</b><br>
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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.
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The ON/OFF ratio with miR-21 is 0.96, which suggested the leakage problem. The regulatory function of the pp21_B toehold switch was not good enough. Thus, pp21_B toehold switch-regulated invertase cannot be well controlled by the miR-21.
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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.
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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.  
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===References===
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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
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 +
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
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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
 +
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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
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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
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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
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7. Shue Wang, Nicholas J Emery, Allen P Liu. A Novel Synthetic Toehold Switch for microRNA Detection in Mammalian Cells. ACS Synthetic Biology 2019; 8 (5): 1079-1088.  
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3431001 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3431007 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K3431001 parameters</partinfo>
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<partinfo>BBa_K3431007 parameters</partinfo>
 
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Revision as of 17:19, 25 October 2020


zr31 Toehold Switch for miR-31 Detection

Introduction

pp21_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 pp21_B toehold switch, we adopted the loop and the linker structure from Wang et al., 2019[7]. 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

















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 RightestTM 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 pp21_B 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 0.96, which suggested the leakage problem. The regulatory function of the pp21_B toehold switch was not good enough. Thus, pp21_B toehold switch-regulated invertase cannot be well 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. Shue Wang, Nicholas J Emery, Allen P Liu. A Novel Synthetic Toehold Switch for microRNA Detection in Mammalian Cells. ACS Synthetic Biology 2019; 8 (5): 1079-1088.

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]