Difference between revisions of "Part:BBa K3431005"

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op21_A 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.
 
op21_A 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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===Model===
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===Design===
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The design of the toehold switch was mainly based on the previous research. 1 2 3 4 5 6 For the op21_A toehold switch, we adopted the loop structure from
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Green et al., 2014 7, and the linker structure is 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)
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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/e/e3/T--CSMU_Taiwan--op21_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/e/e3/T--CSMU_Taiwan--op21_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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<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/e/e4/T--CSMU_Taiwan--op21_A_Ve.png" style="display: block;margin-left: auto;margin-right: auto; width: 100%">
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VIENNA RNA PACKAGE <br>
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<figcaption style="text-align: center;">
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Figure 2. ViennaRNA Package result
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<div style="width=100%; display:flex; align-items: center; justify-content: center;">
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</figcaption>
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<img src="https://static.igem.org/mediawiki/parts/e/e4/T--CSMU_Taiwan--op21_A_Ve.png" style="width:50%;">
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</figure>
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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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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
  
 
===Experiment result===
 
===Experiment result===

Revision as of 12:01, 25 October 2020


op21_A Toehold Switch for miR-21 Detection

Introduction

op21_A 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 op21_A toehold switch, we adopted the loop structure from Green et al., 2014 7, and the linker structure is 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

















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. 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.

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]