Difference between revisions of "Part:BBa K3431035"
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<partinfo>BBa_K3431035 short</partinfo>
 
<partinfo>BBa_K3431035 short</partinfo>
  
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===Description===
 
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.
 
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.
  
 
For this particular toehold switch (zr146_B), we incorporate the loop structure from Pardee, K. et al. and the random linker structure generated by our team.<br>
 
For this particular toehold switch (zr146_B), we incorporate the loop structure from Pardee, K. et al. and the random linker structure generated by our team.<br>
  
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===Model===
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<html>
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<br>
 
NUPACK ANALYSIS <br>
 
NUPACK ANALYSIS <br>
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https://static.igem.org/mediawiki/parts/7/7d/T--CSMU_Taiwan--zr146_B_NU.png
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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/7/7d/T--CSMU_Taiwan--zr146_B_NU.png" style="width:50%;">
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</div>
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<br>
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</html>
 
VIENNA RNA PACKAGE <br>
 
VIENNA RNA PACKAGE <br>
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https://static.igem.org/mediawiki/parts/1/1a/T--CSMU_Taiwan--zr146_B_Ve.png
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<html>
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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/1/1a/T--CSMU_Taiwan--zr146_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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Link to our model page: https://2020.igem.org/Team:CSMU_Taiwan/Model
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===Experiment result===
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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/e1/T--CSMU_Taiwan--zr146_B_%28BBa_K3431044%29.png" style="width:50%">
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</div>
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<br>
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</html>
  
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References:
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===References===
 
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.
 
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.
 
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.
 
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.

Revision as of 17:04, 24 October 2020


zr146_B Toehold Switch for miR-146 Detection

Description

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.

For this particular toehold switch (zr146_B), we incorporate the loop structure from Pardee, K. et al. and the random linker structure generated by our team.

Model


NUPACK ANALYSIS


VIENNA RNA PACKAGE

Link to our model page: https://2020.igem.org/Team:CSMU_Taiwan/Model

Experiment result



References

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. 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.

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]