Difference between revisions of "Part:BBa K3431001"

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<partinfo>BBa_K3431001 short</partinfo>
 
<partinfo>BBa_K3431001 short</partinfo>
  
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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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=== Introduction ===
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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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blblbl
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===Design===
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For this particular toehold switch (pp21), 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 are based.
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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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References:
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<html>
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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/3/31/T--CSMU_Taiwan--pp21_B_NU.png" style="display: block;margin-left: auto;margin-right: auto; width: 70%">
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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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</div>
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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>
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</html>
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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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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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===Characterization using invertase===
  
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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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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”.  
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<html>
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<br>
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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/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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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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</figcaption>
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</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).
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<br>
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</html>
  
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<b>Results</b><br>
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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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===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
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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
 +
 +
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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===Information contributed by City of London UK (2021)===
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[[File:ToeholdTools.png|x200px|center]]
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This toehold switch was characterized <i>in silico</i> using the ToeholdTools project that our team developed.
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See https://github.com/lkn849/thtools for more information.
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Metadata:
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*'''Group:''' City of London UK 2021
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*'''Author:''' Lucas Ng
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*'''Summary:''' Used our software ToeholdTools to investigate the target miRNA specificity and activation of this part.
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Raw data:
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*[[Media:BBa_K3431001_thtest.txt]]
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*[[Media:BBa_K3431001_crt.txt]]
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This contribution was autogenerated by the script '''contrib.py''', available at https://github.com/lkn849/thtools/tree/master/registry.
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----
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This switch was designed to detect the miRNA hsa-miR-21-5p at a temperature of 37°C.
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We tested it against every mature <i>Homo sapiens</i> miRNA in miRBase and our analysis shows that at this temperature it is best used to detect hsa-miR-7156-5p.
 +
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With hsa-miR-7156-5p at 37°C, the switch has a specificity of 14 ± Infinity % and an activation of 0.2 ± 0.9 %.
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These values represent 95% confidence limits (z=1.96).
 +
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The temperature&ndash;activation&ndash;specificity relationship is shown here.
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CRT is an acronym for CelsiusRangeTest, the class in our Python library responsible for the following graph:
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[[File:BBa_K3431001_graph.png|500px|center]]
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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.
 +
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'''Caveats:'''
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*As per the above, we cannot confirm that this switch accurately detects the desired miRNA sequence.
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*The miRNA most targeted by this switch heavily fluctuates based on temperature.Therefore, we cannot confirm the reliability of this switch.
 +
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We do not recommend this part for future usage.
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 11:56, 9 October 2021


pp21_B Toehold Switch for miR-21 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.

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-7156-5p.

With hsa-miR-7156-5p at 37°C, the switch has a specificity of 14 ± Infinity % and an activation of 0.2 ± 0.9 %. 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 K3431001 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.

Caveats:

  • As per the above, we cannot confirm that this switch accurately detects the desired miRNA sequence.
  • The miRNA most targeted by this switch heavily fluctuates based on temperature.Therefore, we cannot confirm the reliability of this switch.

We do not recommend this part for future usage. 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]