Difference between revisions of "Part:BBa K3905004"

Latest revision as of 18:55, 21 October 2021

gen2 517-5p Toehold Switch

Labelled toehold switch in its 'off' state.

This Toehold Switch upregulates the translation of a reporter protein in the CDS downstream in the presence of miRNA-517-5p- an miRNA upregulated in preeclampsia patients. We characterised this switch to show it can discriminate between homologs and in a 4:1 ratio of concentrations, corresponding to the fourfold increase in concentration indicative of the condition

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

Toehold switches contain multiple parts, namely: the trigger binding site, the ribosome binding site (RBS), the seven amino acid long linker sequence and the protein coding sequence.

The trigger binding site on each toehold switch is the reverse complement to the trigger RNA strand. As the miRNAs are both 22 nucleotides long, the trigger site is also 22 nucleotides long. The trigger binding site makes up the lower stem in the ‘off’ state as well as part of the toehold base. We were advised to ensure the trigger site was >10 nucleotides long by Dr Wooli.

When few microRNAs are present, the ribosome binding site of an mRNA coding for luciferase is folded up, so the ribosome cannot unzip the long stem and move down the strand to the start codon to initiate translation.

However, when a microRNA binds to the trigger site, the switch unfolds, decreasing the length of the stem, and exposing the start codon, so translation of luciferase - a reporter protein - can begin.

This was designed using a python script taken from the NUPACK API, there is more information about this here: https://2021.igem.org/Team:City_of_London_UK/Software .

Characterisation

Methodology

For this 517-5p gen2 switch, we tested, at a constant DNA concentration, the target miRNA, miR-517-5p at 9M and 2.25M to replicate the roughly 4-fold increase of expression levels in patients with preeclampsia, as well as a homolog for the miRNA which was miR-518f-5p, this was determined to be the miRNA that has the highest probability of activating the switch aside from the target miRNA itself by our software tool (see software page https://2021.igem.org/Team:City_of_London_UK/Software). We then incubated the DNA, miRNAs with a plasmid containing a gene coding for T7 polymerase (to promote transcription of our toehold switch mRNA) 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 four measurements after D-luciferin in a buffer was injected, at different times.

Results

Line graph comparing the luminescence against the concentration and species of miRNA over time.

The results show a brief, high peak in luminescence for a high concentration of miR-517-5p. This is because, during the incubation time, more luciferase is produced due to an increased rate of translation, so there is a higher concentration of luciferase, so more luciferase-luciferin complexes form at the beginning, converting luciferin to oxyluciferin, producing luminescence.

Graph comparing the luminescence against the concentration and specie of miRNA.

Graph comparing the luminescence against the concentration and species of miRNA in respect to the negative control.

The results show that, compared to the negative control, the homolog ‘miR-518f-5p’ activates the switch more, causing a higher peak, sooner on, and there is only a small increase in luminescence between the control and the target RNA at reduced concentration. Based on this data, we decided that our kit should detect luminescence at some point between 100 and 200 seconds. These diagrams show how the toehold switch can discriminate clearly between miR-517-5p at 9M and 2.25M.

Conclusion

The gen2 517-5p toehold switch causes a noticeable increase in translation of luciferin with increased miRNA concentration, and, with software, they would be able to binarily discriminate between levels of miRNA at concentrations indicative of the condition, given they are above 2.25M in ‘normal’ patients.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal XbaI site found at 15
Illegal XbaI site found at 53
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal XbaI site found at 15
Illegal XbaI site found at 53
25
INCOMPATIBLE WITH RFC[25]
Illegal XbaI site found at 15
Illegal XbaI site found at 53
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3905004 short</partinfo>
-<centre>
 <html>
+<centre>
 <figure>
 <img width="600px" src="https://2021.igem.org/wiki/images/thumb/d/d6/T--City_of_London_UK--Toehold_Switch1.png/1200px-T--City_of_London_UK--Toehold_Switch1.png">
 </figure>
+</centre>
 <figcaption>
 <i>
@@ Line 14: / Line 16: @@
 </figcaption>
 </html>
-<centre>
-<h4>This Toehold Switch upregulates the translation of a reporter protein in the CDS downstream in the presence of miRNA-517-5p- an miRNA upregulated in preeclampsia patients. We characterised this switch to show it can discriminate between homologs and in a 4:1 ration of concentrations, corresponding to the fourfold increase in concentration indicative of the condititon </h4>
+<h4>This Toehold Switch upregulates the translation of a reporter protein in the CDS downstream in the presence of miRNA-517-5p- an miRNA upregulated in preeclampsia patients. We characterised this switch to show it can discriminate between homologs and in a 4:1 ratio of concentrations, corresponding to the fourfold increase in concentration indicative of the condition </h4>
+<html>
+<figure>
+<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">
+</figure>
+<figcaption>
+<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]
+<br><br>
+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>
+</figcaption>
+</html>
 <h2>Functionality</h2>
 Toehold switches contain multiple parts, namely: the trigger binding site, the ribosome binding site (RBS), the seven amino acid long linker sequence and the protein coding sequence.
@@ Line 27: / Line 42: @@
 However, when a microRNA binds to the trigger site, the switch unfolds, decreasing the length of the stem, and exposing the start codon, so translation of luciferase - a reporter protein - can begin.
-This was designed using a python script taken from the NUPACK API, there is more information about this here https://2021.igem.org/Team:City_of_London_UK/Software .
+This was designed using a python script taken from the NUPACK API, there is more information about this here:  https://2021.igem.org/Team:City_of_London_UK/Software .
 <h2>Characterisation</h2>
 <h3>Methodology</h3>
-For this 517-5p gen2 switch, we tested, at a constant DNA concentration, the target miRNA, miR-517-5p at 9M and 2.25M to replicate the roughly 4-fold increase of expression levels in patients with preeclampsia  1 2, as well as a homolog for each miRNA (miR-518f-5p for miR-517-5p), which were determined to be the miRNAs have the highest probability of activating the switches aside from the target miRNAs themselves by our software tool (see software page). We then incubated the DNA, 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 four measurements after D-luciferin in a buffer was injected, at different times.
+For this 517-5p gen2 switch, we tested, at a constant DNA concentration, the target miRNA, miR-517-5p at 9M and 2.25M to replicate the roughly 4-fold increase of expression levels in patients with preeclampsia, as well as a homolog for the miRNA which was miR-518f-5p, this was determined to be the miRNA that has the highest probability of activating the switch aside from the target miRNA itself by our software tool (see software page https://2021.igem.org/Team:City_of_London_UK/Software). We then incubated the DNA, miRNAs with a plasmid containing a gene coding for T7 polymerase (to promote transcription of our toehold switch mRNA) 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 four measurements after D-luciferin in a buffer was injected, at different times.
 <h3>Results</h3>
@@ Line 65: / Line 80: @@
 <figcaption>
 <i>
-Graph comparing the luminescence against the concentration and specie of miRNA in respect to the negative control.
+Graph comparing the luminescence against the concentration and species of miRNA in respect to the negative control.
 </i>
 </figcaption>
@@ Line 72: / Line 87: @@
-The results show that, compared to the negative control, the homolog ‘miR-518f-5p’ activates the switch more, causing an higher peak, sooner on, and there is only a small increase in luminescence between the control and the target RNA at reduced concentration. Based on this data, we decided that our kit should detect luminescence at some point between 100 and 200 seconds.
+The results show that, compared to the negative control, the homolog ‘miR-518f-5p’ activates the switch more, causing a higher peak, sooner on, and there is only a small increase in luminescence between the control and the target RNA at reduced concentration.
+Based on this data, we decided that our kit should detect luminescence at some point between 100 and 200 seconds.
 These diagrams show how the toehold switch can discriminate clearly between miR-517-5p at 9M and 2.25M.
 <h3> Conclusion </h3>
-The gen2 517-5p toehold switch cause a noticeable increase in translation of luciferin with increased miRNA concentration, and, with software, they would be able to binarily discriminate between levels of miRNA at concentrations indicative of the condition, given they are above 2.25M in ‘normal’ patients.
+The gen2 517-5p toehold switch causes a noticeable increase in translation of luciferin with increased miRNA concentration, and, with software, they would be able to binarily discriminate between levels of miRNA at concentrations indicative of the condition, given they are above 2.25M in ‘normal’ patients.
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