Difference between revisions of "Part:BBa K3431013"
 
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<partinfo>BBa_K3431013 short</partinfo>
 
<partinfo>BBa_K3431013 short</partinfo>
  
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This toehold switch has been designed to open up its hairpin loop structure upon binding with miRNA-146, 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-146, we optimise the toehold switch structure by altering their loop region and linker sequence. We incorporate two designs of the loop region from two articles: the original work on toehold switch (Green, A.A. et al., 2014) and the adaptation of toehold switch to detect zika virus (Pardee, K. et al., 2016). Pardee, K. et al. have truncated the loop structure from 19 base pairs in the original work conducted by Green, A.A. et al. to 12 base pairs in order to reduce the leakage of output expression. Hence we hope to observe an increase in the output's dynamic range by implementing the loop sequence utilised by Pardee, K. et al.. As for our selection on the linker sequences, we choose to test out the linker sequence from Pardee, K. et al. and a random linker sequence which we generated in order to minimize the free energy of toehold switch mRNA secondary structure.
 
  
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For this particular toehold switch (or146_A), we incorporate the loop structure from Green, A.A. et al. and the random linker structure generated by our team.
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===Introduction===
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or146_A toehold switch is a regulatory part for the downstream reporter gene. With this part, the protein expression can be controlled by the miR-146. 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-146, 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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References:
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===Design===
  
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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 design of the toehold switch was mainly based on the previous research<sup>[1][2][3][4][5][6]</sup>. For the or146_A toehold switch, we adopted the loop structure from Green et al., 2014<sup>[7]</sup>, and the linker structure is the random linker design by iGEM_CSMU_2020. 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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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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<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/3/33/T--CSMU_Taiwan--or146_A_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/7/78/T--CSMU_Taiwan--or146_A_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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Error bars represent the standard deviation.
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The line of best fit was calculated using a univariate cubic spline weighted inverse to each point's standard error.
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===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”.
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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/e6/T--CSMU_Taiwan--or146_A_%28BBa_K3431029%29.png" style="width:40%">
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</div>
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Figure. 3. The glucose productions of the or146_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-146 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-146 is 2.41, which suggested the regulatory function of the toehold switch. By contrast, the ON/OFF ratios with miR-191 and miR-223 are 1.70 and 1.71, respectively. These ratios are close to 1, meaning the or146_A toehold switch has high specificity. As a result, or146_A_ToeholdSwitch-Regulated Invertase has been proven to be useful for miR-146 detection.
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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. Green AA, Silver PA, Collins JJ, Yin P. Toehold switches: de-novo-designed regulators of gene expression. Cell 2014; 159(4): 925-39.
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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_K3431013_thtest.txt]]
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*[[Media:BBa_K3431013_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-146a-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-146a-5p.
 +
 
 +
With hsa-miR-146a-5p at 37°C, the switch has a specificity of 18 ± Infinity % and an activation of 3 ± 3 %.
 +
These values represent 95% confidence limits (z=1.96).
 +
 
 +
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:
 +
 
 +
[[File:BBa_K3431013_graph.png|500px|center]]
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 23:33, 12 October 2021


or146_A Toehold Switch for miR-146 Detection


Introduction

or146_A toehold switch is a regulatory part for the downstream reporter gene. With this part, the protein expression can be controlled by the miR-146. 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-146, 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 or146_A toehold switch, we adopted the loop structure from Green et al., 2014[7], and the linker structure is the random linker design by iGEM_CSMU_2020. 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

















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.

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 or146_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-146 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-146 is 2.41, which suggested the regulatory function of the toehold switch. By contrast, the ON/OFF ratios with miR-191 and miR-223 are 1.70 and 1.71, respectively. These ratios are close to 1, meaning the or146_A toehold switch has high specificity. As a result, or146_A_ToeholdSwitch-Regulated Invertase has been proven to be useful for miR-146 detection.

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.

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-146a-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-146a-5p.

With hsa-miR-146a-5p at 37°C, the switch has a specificity of 18 ± Infinity % and an activation of 3 ± 3 %. 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 K3431013 graph.png

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 9
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 9
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 9
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 9
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 9
  • 1000
    COMPATIBLE WITH RFC[1000]