Difference between revisions of "Part:BBa K3905006"
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<partinfo>BBa_K3905006 short</partinfo>
 
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This toehold switch can act as a riboregulator to inhibit translation until both micro-RNA 210-3p and 517-5p are present. Both of these microRNAs bind to an anti-miRNA (part BBa_K3905007) which forms a complex that presents both target microRNAs as a trigger to the toehold switch. This triggers the toehold switch to unravel, exposing the RBS and start codon, allowing translation of a protein. This protein should be a reporter protein so that the activity of the toehold switch can be detected and presence of the microRNAs can be confirmed.
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<img width="800px" src="https://2021.igem.org/wiki/images/thumb/e/e3/T--City_of_London_UK--AndGateTHS.png/800px-T--City_of_London_UK--AndGateTHS.png">
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<img width="800px" src="https://2021.igem.org/wiki/images/thumb/6/64/T--City_of_London_UK--Toehold_Switch3.png/800px-T--City_of_London_UK--Toehold_Switch3.png">
 
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Diagram of And-Gate toehold switch in action.  
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<i>Diagram of AND-Gate toehold switch in 'OFF' state.</i>
 
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AND-Gate Toehold Switch
 
  
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Having tested the single trigger switches, and thereby demonstrating how our concept of a toehold switch works as hypothesised, the main purpose of testing the AND-Gate Switch was to see whether it could discriminate between a high concentration of one miRNA, and a low concentration of another, and a high concentration of both. This was in case a patient had increased levels of one miRNA and not the other, which would not be indicative of Preeclampsia.  
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This toehold switch upregulates translation of a CDS downstream in the presence of two specific miRNA triggers - miR-517-5p and miR-210-3p - which are upregulated in patients with preeclampsia. We characterised this switch to show it can discriminate between a 4:1 ratio of concentrations, as a four-fold increase in concentration of both miRNAs is indicative of the condition (see below).
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<html>
 
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<figure>
 
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<img width="800px" src="https://2021.igem.org/wiki/images/thumb/c/cb/T--City_of_London_UK--Graph_8.png/1200px-T--City_of_London_UK--Graph_8.png">
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<img width="800px" src="https://2021.igem.org/wiki/images/thumb/7/75/T--City_of_London_UK--210_517_miRNA_graph.png/800px-T--City_of_London_UK--210_517_miRNA_graph.png">
 
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Graph of luminescence against the volume and type of miRNA inputted
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<i>Reza M. A. N., Kolsoum S. et al., 2019. Quantification of circulating miR-517c-3p and miR-210-3p levels in preeclampsia. Pregnancy Hypertension, Volume 16, 2019, Pages 75-78, ISSN 2210-7789. Available at: https://doi.org/10.1016/j.preghy.2019.03.004 [Accessed 19 October 2021]
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Condrat, C.E. et al., 2020. miRNAs as Biomarkers in Disease: Latest Findings Regarding Their Role in Diagnosis and Prognosis. Cells, 9(2), p.276. Available at: http://dx.doi.org/10.3390/cells9020276. [Accessed 19 October 2021]. </i>
 
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This toehold switch
  
 
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<figure>
 
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<img width="800px" src="https://2021.igem.org/wiki/images/0/0c/T--City_of_London_UK--And_Gate2.png">
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<img width="800px" src="https://2021.igem.org/wiki/images/thumb/e/e3/T--City_of_London_UK--AndGateTHS.png/800px-T--City_of_London_UK--AndGateTHS.png">
 
</figure>
 
</figure>
 
<figcaption>
 
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Graph of luminescence against the volume and type of RNA compared to the negative control.  
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<i>Diagram of And-Gate toehold switch in action. </i>
 
</figcaption>
 
</figcaption>
 
</html>
 
</html>
  
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The graphs show how, compared to the negative control, which shows leaky expression of the toehold switch, the luminescence output of the sample with 9M concentration of both miRNA triggers is 92% higher. Interestingly, the percentage increase in luminescence of the miR-210-3p at 9M and miR-517-5p at 2.25M is twice more than twice as high as the miRNAs at flipped concentrations - 28% compared to 12%. This could be because the miR-210-3p binding site is further downstream than the miR-517-5p binding site, so miR-210-3p is able to bind and partially unfold the switch in the absence of miR-517-5p, increasing the rate of translation. However, as the miR-517-5p binding site is further upstream, it is completely bound up in hydrogen bonds, so it is not accessible to the miRNA unless it binds as the switch is being transcribed.
 
  
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AND-Gate Toehold Switch
 +
 +
Having tested the single trigger switches, and thereby demonstrating how our concept of a toehold switch works as hypothesised, the main purpose of testing the AND-Gate Switch was to see whether it could discriminate between a high concentration of one miRNA, and a low concentration of another, and a high concentration of both. This was in case a patient had increased levels of one miRNA and not the other, which would not be indicative of Preeclampsia.
 
<html>
 
<html>
 
<figure>
 
<figure>
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<img width="800px" src="https://2021.igem.org/wiki/images/0/0c/T--City_of_London_UK--And_Gate2.png">
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<img width="400px" src="https://2021.igem.org/wiki/images/thumb/7/75/T--City_of_London_UK--210_517_miRNA_graph.png/800px-T--City_of_London_UK--210_517_miRNA_graph.png">
 
</figure>
 
</figure>
 
<figcaption>
 
<figcaption>
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3D diagram of And-Gate toehold switch with all parts bound.
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<i>Graph of luminesce against the volume and type of miRNA inputted</i>
 
</figcaption>
 
</figcaption>
 
</html>
 
</html>
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The graphs show how, compared to the negative control, which shows leaky expression of the toehold switch, the luminescence output of the sample with 9M concentration of both miRNA triggers is 92% higher. Interestingly, the percentage increase in luminescence of the miR-210-3p at 9M and miR-517-5p at 2.25M is twice more than twice as high as the miRNAs at flipped concentrations - 28% compared to 12%. This could be because the miR-210-3p binding site is further downstream than the miR-517-5p binding site, so miR-210-3p is able to bind and partially unfold the switch in the absence of miR-517-5p, increasing the rate of translation. However, as the miR-517-5p binding site is further upstream, it is completely bound up in hydrogen bonds, so it is not accessible to the miRNA unless it binds as the switch is being transcribed.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Revision as of 16:24, 21 October 2021


gen3 210-3p-517-5p AND-Gate Toehold Switch


Diagram of AND-Gate toehold switch in 'OFF' state.


This toehold switch upregulates translation of a CDS downstream in the presence of two specific miRNA triggers - miR-517-5p and miR-210-3p - which are upregulated in patients with preeclampsia. We characterised this switch to show it can discriminate between a 4:1 ratio of concentrations, as a four-fold increase in concentration of both miRNAs is indicative of the condition (see below).


Reza M. A. N., Kolsoum S. et al., 2019. Quantification of circulating miR-517c-3p and miR-210-3p levels in preeclampsia. Pregnancy Hypertension, Volume 16, 2019, Pages 75-78, ISSN 2210-7789. Available at: https://doi.org/10.1016/j.preghy.2019.03.004 [Accessed 19 October 2021]

Condrat, C.E. et al., 2020. miRNAs as Biomarkers in Disease: Latest Findings Regarding Their Role in Diagnosis and Prognosis. Cells, 9(2), p.276. Available at: http://dx.doi.org/10.3390/cells9020276. [Accessed 19 October 2021].


This toehold switch

Diagram of And-Gate toehold switch in action.


AND-Gate Toehold Switch

Having tested the single trigger switches, and thereby demonstrating how our concept of a toehold switch works as hypothesised, the main purpose of testing the AND-Gate Switch was to see whether it could discriminate between a high concentration of one miRNA, and a low concentration of another, and a high concentration of both. This was in case a patient had increased levels of one miRNA and not the other, which would not be indicative of Preeclampsia.

Graph of luminesce against the volume and type of miRNA inputted


The graphs show how, compared to the negative control, which shows leaky expression of the toehold switch, the luminescence output of the sample with 9M concentration of both miRNA triggers is 92% higher. Interestingly, the percentage increase in luminescence of the miR-210-3p at 9M and miR-517-5p at 2.25M is twice more than twice as high as the miRNAs at flipped concentrations - 28% compared to 12%. This could be because the miR-210-3p binding site is further downstream than the miR-517-5p binding site, so miR-210-3p is able to bind and partially unfold the switch in the absence of miR-517-5p, increasing the rate of translation. However, as the miR-517-5p binding site is further upstream, it is completely bound up in hydrogen bonds, so it is not accessible to the miRNA unless it binds as the switch is being transcribed.

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 82