Results
This section details the preparation of pET-14b-EGFP vectors, cloning of Toehold Switch constructs, validation by DNA sequencing, and reporter expression assays for binding toehold switches to miRNAs.
Preparation of pET-14b-EGFP Vectors
Linearization of pET-14b-EGFP Plasmids by Ncol and Ndel Restriction Digestion
We amplified the purchased pET-14b-EGFP plasmids by transforming competent DH5α E. coli cells using the heat shock method. After introducing the plasmid, we grew the cells on LB plates with ampicillin to select for successful transformants. Once colonies grew, we extracted the plasmid DNA using a mini-prep procedure.
The digestion of the pET-14b-EGFP vector was performed using the following materials:
- Plasmid DNA: 1 μg
- 10X Restriction Buffer: 5 μL
- Ncol-HF: 1 μL
- Ndel: 1 μL
- DNase-free water: add to a final volume of 50 μL
The above gel electrophoresis demonstrates that under different forms of the plasmid, the plasmids travelled at different speeds under gel electrophoresis. This is essential for separating the digested plasmid from the undigested ones. It is because when digested, the plasmid would change from its supercoiled form, which travelled faster in gel due to higher compactness, to its linearised form which travelled slower in gel.
Cloning of Toehold Switch Constructs
Transformation of Recombinant Plasmids
Unsuccessful Attempts
Attempt 1: After ligating the pET-14b-EGFP plasmid with Toehold Switch 1 and Toehold Switch 2, we transformed E. coli with the recombinant plasmids: Toehold Switch 1 plasmids (T1 plasmids) and Toehold Switch 2 plasmids (T2 plasmids). The bacteria were then cultured on agar plates containing LB medium and ampicillin. We also set up two controls: a positive control with bacteria transformed with the pET-14b-EGFP plasmid (non-recombinant) and a negative control with no bacteria.
Results
Interpretation and conclusion
The result of negative control indicated that the plate and antibiotic were functioning correctly, without any contamination. The positive control confirmed that the transformation procedure was successful, as the bacteria expressed the antibiotic resistance gene, allowing them to survive on the ampicillin plates.
We observed one colony of bacteria on each agar plate transformed with T1 and T2 plasmids, suggesting that the transformation of bacteria with the Toehold Switch plasmids may have been successful. Since ampicillin selection alone cannot confirm the presence of recombinant clones, we picked the colonies and sent them for DNA sequencing.
However, the DNA sequencing results showed that both Toehold Switches 1 and 2 were not ligated into the plasmids; the base sequences of the plasmids remained unchanged, and we could not find the sequences of Toehold Switches 1 and 2 between NcoI and NdeI.
Due to limitations in our laboratory skills for gel extraction and plasmid purification, we decided to make another attempt.
Attempt 2: We restarted the experiment with some modifications: we extracted as much gel as possible, aimed to maximize the concentration of the plasmids and proper mixing of inserts into vectors. We then carried out the transformation of bacteria with the T1 and T2 plasmids once again. The results are shown in the following figures.
Results
Interpretation and conclusion
There was one colony of bacteria transformed with T2 plasmids on the agar plate. We picked the colony and sent it to DNA sequencing. Unfortunately, the sequencing result showed that the insertion of Toehold Switch 2 into the plasmid failed, as the base sequence of the plasmid remained unchanged and the base sequences of Toehold switch 2 cannot be found between NcoI and NdeI. The result of DNA sequencing shows that the ligation was unsuccessful. This can be caused by the low concentration of plasmids (vectors) or the ratio of insert to vector.
Attempt 3: This time, we used maxiprep instead of miniprep to obtain a higher concentration of plasmid (the vector). We added the 5’ phosphate group to oligos of the Toehold Switches for insertion. Then we carried out ligation and transformation again. To increase our chances of success, we used another ratio of concentration of the plasmid (the vector) to the Toehold Switches 1 and 2 (the inserts), which was 1:10. After ligation, we did the transformation. Then we cultured the transformed bacteria in LB medium with ampicillin.
Results
Interpretation and conclusion
Only bacteria transformed with T1 Plasmids could be cultured. Bacteria were picked up and were sent for DNA sequencing.
However, the result of DNA sequencing showed that the Toehold Switch 1 failed to be inserted into the plasmids. We had to revise our protocol.
Successful Cloning
We used a high insert-to-vector ratio (20:1) to ligate toehold switches 7, 8, and 9 into pET-14b-EGFP plasmids extracted via maxiprep. The resulting recombinant plasmids were transformed into E. coli and plated on LB agar containing ampicillin for colony growth.
Results:
Interpretation and conclusion
The negative control verified that the antibiotic was effective and that no contamination occurred during bacterial culture.
The positive control confirmed that E. coli transformed with plasmid vectors with intact ampicillin resistance gene could successfully grow.
Colonies observed on agar plates with E. coli transformed with T8 plasmids suggested cloning of T8 may be successful. However, since ampicillin selection alone cannot confirm recombinant clones, we verified the T8 recombinant plasmids through DNA sequencing.
Validation by DNA Sequencing
The DNA sequencing results confirmed that our E.coli colonies were actually transformed with our toehold switch 8 recombinant plasmids, proving our success in cloning.
Reporter Expression
Binding of toehold switches 1,2,3 to miRNA-92a-3p; toehold switches 7,8,9 to miRNA-135b-5p
Attempt 1 (Preliminary test): Cell free expression of Toehold Switch 3 plasmids only
To test the binding of toehold switches, we used an in vitro expression (IVE) kit, which allows the transcription and translation of the EGFP gene on plasmids in a cell-free system. We mixed 1.5 µL of 5 µM miRNA samples with 62.5 ng of plasmid, with or without the toehold switches, in a total reaction volume of 6 µL. The samples were then observed under blue light. Green fluorescence would indicate successful binding of the miRNA to the toehold switches, leading to the opening of the hairpin structure and subsequent expression of EGFP.
A negative control, containing no plasmid, and a positive control, containing the non-recombinant plasmid, were also included. The table below shows the contents of the tubes in the IVE kit and shows the contents of 6 tubes in in vitro expression.
As minimal differences were found between the positive and negative controls under influence of blue light, we found that it was necessary to use a plate reader for measuring the fluorescence intensity. Also, we had to optimize the amount of plasmids we used in the in vitro expression kit.
Optimisation of Positive Control
In order to optimize the amount of plasmids used in the in vitro expression kit, a total of 3 trials have been performed.
Trial 1
Content of the tubes in IVE Kit
Measurement of fluorescence intensity in tubes with different amount of plasmids
Optimisation: Trial 2 and 3
Trial 2
Measurement of fluorescence intensity in tubes with different amount of plasmids
Trial 3
Measurement of fluorescence intensity in tubes with different amount of plasmids
Average fluorescence intensity in tubes with different amount of plasmids
When comparing the fluorescence intensity across different amounts of plasmids, the highest fluorescence was observed with 7.5 ng of plasmids. Therefore, 7.5 ng was identified as the optimal amount for subsequent experiments on in vitro expression.
Attempt 2: Cell free expression of Toehold Switch plasmids
The table below shows the contents of each tube in the IVE kit. Tubes 1 to 5 did not contain any target miRNA, while tubes 6 to 10 included 5 µM of the target miRNA (miRNA-92a-3p). The negative control was the tube without any plasmids, whereas the positive control contained the plasmid pET-14b-EGFP.
Content of the tubes used in in vitro Expression
Tubes 1 and 6 serve as the negative controls: These ensure that any observed fluorescence results specifically from the interaction of the miRNA with the toehold switches, rather than from other sources. Since no plasmid is present, any fluorescence in these controls suggests possible contamination or nonspecific reactions occurring within the kit.
Tubes 5 and 10 serve as the positive controls: They confirm that the experimental conditions are suitable for the desired reaction. With the non-recombinant plasmid present, the observation of green fluorescence indicates that the components of the IVE kit and the overall experimental setup are functioning correctly.
The table below shows the fluorescence intensity of each tube in in vitro expression.
*PBS refers to the tube with Phosphate-buffered saline (PBS) only. PBS is a buffer solution (pH ~ 7.4) commonly used in biological research. All the samples were made up to 100 µL by adding PBS before measurement of fluorescence, so It serves for measuring the background fluorescence.
Based on the data, the fold change of fluorescence intensity (+miRNA / -miRNA) was calculated and presented in the table below.
From the above data, we can see that: Toehold Switch 1 displayed a fold change of 2.12 in fluorescence, indicating an increase upon miRNA addition. This suggests that it can effectively detect miRNA, though it shows slightly less efficacy compared to Toehold Switch 2. Toehold Switch 2 achieved a fold change of 2.25, representing the highest increase in fluorescence among the tested switches for miR-92a-3p. This reinforces its designation as the most effective candidate for miR-92a-3p detection.
Toehold Switch 3 showed a decrease in fluorescence, likely due to issues with binding or structural stability that prevent effective translation. This indicates that it is unsuitable for miRNA detection.
Negative controls (nuclease-free water and setups without Toehold Switches) confirmed that the fluorescence change was due to the interaction of miRNA with the toehold switches, rather than non-specific effects.
Conclusion
Both Toehold Switch 1 and Toehold Switch 2 exhibited sufficient fluorescence after the addition of miRNA, but Toehold Switch 2 was slightly more effective, showing a higher fold change than that of Toehold Switch 1.
While both switches are viable candidates, the slight advantage in Toehold Switch 2 makes it the preferred choice for further investigation.