Difference between revisions of "Part:BBa K3431011"
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<partinfo>BBa_K3431011 short</partinfo>
 
<partinfo>BBa_K3431011 short</partinfo>
  
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===Description===
 
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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 (zr146_A), we incorporate the loop structure from Pardee, K. et al. and the random linker structure generated by our team.
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===Introduction===
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zr146_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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===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 zr146_A toehold switch, we adopted the loop and linker structure from Green et al., 2016<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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===Model===
 
 
<html>
 
<html>
 
<br>
 
<br>
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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/0/00/T--CSMU_Taiwan_--zr146_A_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/0/00/T--CSMU_Taiwan_--zr146_A_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>
 
</div>
 
</div>
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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/7/70/T--CSMU_Taiwan--zr146_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>
 
</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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===Characterization using invertase===
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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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<div style="width=100%; display:flex; align-items: center; justify-content: center;">
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<br>
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<img src="https://static.igem.org/mediawiki/parts/7/70/T--CSMU_Taiwan--zr146_A_Ve.png" style="width:50%;">
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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/2/23/T--CSMU_Taiwan--zr146_A_%28BBa_K3431027%29.png" style="width:40%">
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</div>
 
</div>
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Figure. 3. The glucose productions of the zr146_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).
 
<br>
 
<br>
 
</html>
 
</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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<b>Results</b><br>
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The glucose concentration in the ON state with miR-146 is about 450 mg/dL, indicating the high sensitivity of the toehold switch. The ON/OFF ratio with miR-146 is 2.07, which suggested the regulatory function of the toehold switch. By contrast, the ON/OFF ratios with miR-191 and miR-223 are 1.09 and 1.31, respectively. These ratios are close to 1, meaning the zr146_A toehold switch has high specificity. As a result, zr146_A_ToeholdSwitch-Regulated Invertase has been proven to be useful for miR-146 detection.
 +
===Characterization using invertase===
 +
 
 +
To understand the correlation of the trigger amount and the glucose production, we added different amounts of miR-146 to the protein synthesis kit and produced the proteins at 37℃ for 2 hours. We would then add 5μl of 0.5M sucrose and measured the glucose concentration with the glucose meter after 30 minutes.
 
<html>
 
<html>
 
<br>
 
<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/2/23/T--CSMU_Taiwan--zr146_A_%28BBa_K3431027%29.png" style="width:50%">
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<img src="https://static.igem.org/mediawiki/parts/4/40/T--CSMU_Taiwan--EXP_5_zr146A.png" style="width:40%">
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</div>
 
</div>
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Figure. 3. The glucose productions of the zr146_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).
 
<br>
 
<br>
 
</html>
 
</html>
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<b>Results</b><br>
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As shown above, the glucose concentration rose as the miR-146 triggers increased, representing a positive correlation.
  
 
===References===
 
===References===
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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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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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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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 +
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
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 +
7. 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.
 
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===Usage and Biology===
 
===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3431011 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_K3431011 parameters</partinfo>
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<partinfo>BBa_K3431007 parameters</partinfo>
 
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Revision as of 16:10, 25 October 2020


zr146_A Toehold Switch for miR-146 Detection


Introduction

zr146_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 zr146_A toehold switch, we adopted the loop and linker structure from Green et al., 2016[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

















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 zr146_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 glucose concentration in the ON state with miR-146 is about 450 mg/dL, indicating the high sensitivity of the toehold switch. The ON/OFF ratio with miR-146 is 2.07, which suggested the regulatory function of the toehold switch. By contrast, the ON/OFF ratios with miR-191 and miR-223 are 1.09 and 1.31, respectively. These ratios are close to 1, meaning the zr146_A toehold switch has high specificity. As a result, zr146_A_ToeholdSwitch-Regulated Invertase has been proven to be useful for miR-146 detection.

Characterization using invertase

To understand the correlation of the trigger amount and the glucose production, we added different amounts of miR-146 to the protein synthesis kit and produced the proteins at 37℃ for 2 hours. We would then add 5μl of 0.5M sucrose and measured the glucose concentration with the glucose meter after 30 minutes.

Figure. 3. The glucose productions of the zr146_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
As shown above, the glucose concentration rose as the miR-146 triggers increased, representing a positive correlation.

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. 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


Assembly Compatibility:
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    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]