Difference between revisions of "Part:BBa K3905010"

Latest revision as of 19:21, 21 October 2021

T7 / gen3 210-3p-517-5p AND-Gate / Luciferase / Double Terminator

Composite Information

This composite part is used for testing the presence of hsa-Micro-RNA-210-3p and 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_K3905006). Placing a reporter protein downstream of this toehold switch part will mean that when our trigger RNAs, miR-210-3p and miR-517-5p are present in high enough quantities, the protein in the CDS 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 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).

Functionality

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.

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.

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

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.

Sequence and Features