Part:BBa_K3905008
T7 / gen2 517-5p Toehold Switch / Luciferase / Double Terminator
Composite Information
This composite part is used for testing the presence of hsa Micro-RNA 517-5p. It has a T7 promoter for expression of functional RNA (Part:BBa_K1614000). This is followed by the code for our toehold switch (Part:BBa_K3905004). Placing a reporter protein downstream of this toehold switch part will mean that when our trigger RNA, miR-517-5p is present in high enough quantities, the protein will be translated and the reporter protein can be observed. Our chosen reporter protein is luciferase (Part:BBa_I712019). When Luciferin is added, luciferase will catalyse a reaction that produces luminescence. This light can be detected with a luminometer to affirm a positive test.
Toehold Switch Information
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
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
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.
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
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