Difference between revisions of "Part:BBa K3905006"
 
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<partinfo>BBa_K3905006 short</partinfo>
 
<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/6/64/T--City_of_London_UK--Toehold_Switch3.png/800px-T--City_of_London_UK--Toehold_Switch3.png">
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<figcaption>
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<i>Diagram of AND-Gate toehold switch in 'OFF' state.</i>
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<h4>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).</h4>
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<figure>
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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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<figcaption>
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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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<br><br>
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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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<h2>Functionality</h2>
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<img width="550px" 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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<figcaption>
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<br>
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<i>Diagram of AND-Gate toehold switch in 'OFF' state.</i>
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</figcaption>
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All of our toehold switches work by repressing translation in the absence of a trigger RNA. As seen in the diagram above, the Ribosome Binding site is upstream and downstream of a long stem. This stem must collapse in order to initiate translation. Typically, the method of collapsing a stem involves a single miRNA or RNA trigger binding to a single trigger site, thereby partially collapsing the stem, such that the ribosome binding site can move down to the start codon from the RBS to initiate translation. However, we wanted to create a switch which could detect multiple miRNA triggers, decreasing the cost of Toehold Switch tests, as only one tube and luminometer is needed to detect two miRNAs, while maintaining the specificity of having multiple triggers.
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<figure>
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<img width="600px" src="https://2021.igem.org/wiki/images/thumb/0/03/T--City_of_London_UK--ComplexFormation.png/800px-T--City_of_London_UK--ComplexFormation.png">
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<figcaption>
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<i>Diagram of Trigger Complex formation.</i>
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Thus, we designed a synthetic anti-miRNA which would bind the two triggers - miR-210-3p and miR-517-5p, creating a complex which itself would act as a trigger.
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<figure>
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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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<i>Diagram of AND-Gate activation mechanism by the Trigger Complex.</i>
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Once the complex has formed, the miR-210-3p section binds to the miR-210-3p binding site, partially unfolding the switch, then the anti-miRNA binds to the anti-miRNA binding site, further unfolding the switch, and finally the miR-517-5p section unfolds the switch fully, such that the RBS is able to initiate translation, typically of a reporter protein.
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<h2>Characterisation</h2>
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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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<h3>Methodology</h3>
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We ordered linear DNA with 250 random base pairs before and after the prefix and suffix respectively on the advice of our TXTL kit supplier. We first suspended our gBlocks, one consisting of a T7 promoter, upstream of the gen3 AND-gate toehold switch, upstream of a firefly luciferase CDS and the other consisting of the anti-miRNA under the control of a T7 Promoter, to reach an overall concentration of 6.7nM in TE buffer.
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We then tested, at a constant DNA concentration, the target miRNAs (miR-517-5p and miR-210-3p) at 9M and 2.25M combinations to replicate the roughly 4-fold increase of expression levels in patients with preeclampsia. We then incubated the DNA for the switch and luciferase, the DNA for the anti-miRNA, miRNAs with a plasmid containing a gene coding for T7 polymerase (to promote transcription of our toehold switch mRNAs) as well as a TXTL cell-free master mix for 1 hour, and then pipetted the contents of each tube into 8 wells of a 96 well plate, and took a measurement after 155s as this was in the region of high expression for high miRNA concentration samples observed in the single trigger switches.
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<h3>Results</h3>
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<html>
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<figure>
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<img width="600px" src="https://2021.igem.org/wiki/images/thumb/c/cb/T--City_of_London_UK--Graph_8.png/800px-T--City_of_London_UK--Graph_8.png
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">
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</figure>
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<figcaption>
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<i>Graph showing the relative luminescence of four three miRNA samples of varied concentration, in addition to a negative control</i>
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</figcaption>
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</html>
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<html>
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<figure>
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<img width="600px" src="https://2021.igem.org/wiki/images/thumb/d/d1/T--City_of_London_UK--Graph_7.png/800px-T--City_of_London_UK--Graph_7.png
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">
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</figure>
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<figcaption>
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<i>Graph showing the percentage increase of luminescence observed of three miRNA samples of varied concentration, compared to the negative control</i>
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</figcaption>
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</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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<h3>Conclusion</h3>
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Based on these results we concluded that our novel AND-gate switch is able to successfully discriminate between concentrations of miRNAs at 4:1 concentrations, given the lower concentration is greater than 2.25M - hence why we employed an isothermal amplification method in our project.
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<h2>Improvement of Existing Parts</h2>
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Previously, toehold switches were only able to detect a single RNA strand at a time. Many teams in the past have submitted toehold switches with only one input such as the 2017 Batman project (Part:BBa_K2206000). This could prove problematic if multiple strands need to be detected as it is difficult to monitor and create the hardware to help detect a conglomerate of RNA. Furthermore, switches which induce a reporter protein in a CDS are unspecific. This is because the same miRNAs are upregulated in multiple conditions as extra-cellular microRNA networks are complex. To combat this issue, we developed an AND-gate switch which can detect two RNAs simultaneously. The ability to detect two RNAs for one condition means that luciferase is only produced when two trigger RNAs are present at sufficient concentrations which helps increase the specificity.  It is not inconceivable to think that in the near future other teams will be able to scale up our novel design to produce systems that are able to detect several microRNAs. This would exponentially increase specificity to a condition.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 01:05, 22 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].


Functionality



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


All of our toehold switches work by repressing translation in the absence of a trigger RNA. As seen in the diagram above, the Ribosome Binding site is upstream and downstream of a long stem. This stem must collapse in order to initiate translation. Typically, the method of collapsing a stem involves a single miRNA or RNA trigger binding to a single trigger site, thereby partially collapsing the stem, such that the ribosome binding site can move down to the start codon from the RBS to initiate translation. However, we wanted to create a switch which could detect multiple miRNA triggers, decreasing the cost of Toehold Switch tests, as only one tube and luminometer is needed to detect two miRNAs, while maintaining the specificity of having multiple triggers.



Diagram of Trigger Complex formation.


Thus, we designed a synthetic anti-miRNA which would bind the two triggers - miR-210-3p and miR-517-5p, creating a complex which itself would act as a trigger.



Diagram of AND-Gate activation mechanism by the Trigger Complex.


Once the complex has formed, the miR-210-3p section binds to the miR-210-3p binding site, partially unfolding the switch, then the anti-miRNA binds to the anti-miRNA binding site, further unfolding the switch, and finally the miR-517-5p section unfolds the switch fully, such that the RBS is able to initiate translation, typically of a reporter protein.


Characterisation

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.


Methodology

We ordered linear DNA with 250 random base pairs before and after the prefix and suffix respectively on the advice of our TXTL kit supplier. We first suspended our gBlocks, one consisting of a T7 promoter, upstream of the gen3 AND-gate toehold switch, upstream of a firefly luciferase CDS and the other consisting of the anti-miRNA under the control of a T7 Promoter, to reach an overall concentration of 6.7nM in TE buffer.

We then tested, at a constant DNA concentration, the target miRNAs (miR-517-5p and miR-210-3p) at 9M and 2.25M combinations to replicate the roughly 4-fold increase of expression levels in patients with preeclampsia. We then incubated the DNA for the switch and luciferase, the DNA for the anti-miRNA, miRNAs with a plasmid containing a gene coding for T7 polymerase (to promote transcription of our toehold switch mRNAs) as well as a TXTL cell-free master mix for 1 hour, and then pipetted the contents of each tube into 8 wells of a 96 well plate, and took a measurement after 155s as this was in the region of high expression for high miRNA concentration samples observed in the single trigger switches.


Results



Graph showing the relative luminescence of four three miRNA samples of varied concentration, in addition to a negative control



Graph showing the percentage increase of luminescence observed of three miRNA samples of varied concentration, compared to the negative control


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.


Conclusion

Based on these results we concluded that our novel AND-gate switch is able to successfully discriminate between concentrations of miRNAs at 4:1 concentrations, given the lower concentration is greater than 2.25M - hence why we employed an isothermal amplification method in our project.


Improvement of Existing Parts

Previously, toehold switches were only able to detect a single RNA strand at a time. Many teams in the past have submitted toehold switches with only one input such as the 2017 Batman project (Part:BBa_K2206000). This could prove problematic if multiple strands need to be detected as it is difficult to monitor and create the hardware to help detect a conglomerate of RNA. Furthermore, switches which induce a reporter protein in a CDS are unspecific. This is because the same miRNAs are upregulated in multiple conditions as extra-cellular microRNA networks are complex. To combat this issue, we developed an AND-gate switch which can detect two RNAs simultaneously. The ability to detect two RNAs for one condition means that luciferase is only produced when two trigger RNAs are present at sufficient concentrations which helps increase the specificity. It is not inconceivable to think that in the near future other teams will be able to scale up our novel design to produce systems that are able to detect several microRNAs. This would exponentially increase specificity to a condition.

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