1. Gel electrophoresis (Collaborated with Team HKU)
With the help of HKU team, we are able to get the gel electrophoresis to analyse whether our design was actually assembled. As seen inthe first gel result. All 8 oligos come together as the shift observed in the last lane.
To further prove that our nano-cube is formed as per our design, we conducted another gel electrophoresis, with each of the samples containing two oligos, which should not be binded together. As shown above, each of the samples, except of the nano-cube, contains two distinct bands, indicating that these two oligos did not bind together, which further proves our design.
Also, the band for the target could not be found when nano-cube and target were placed together (the last lane), indicating that the target should be successfully bound with the nano-cube.
All in all, these two gel results suggested that our nano-cube should be formed as per our design.
2. DNA Peroxidase Assay Result
Our results are shown above. Figure 7a showed that the nano-cube gave a very high signal of absorbance at 0.85 on its own. The presence of target showed a low signal at 0.75. We then conducted a control experiment with random DNA added to the cube. Figure 7b suggested that no reduction of absorbance was recorded, indicating that our nano-cube is specific to H3N2 virus. Then, we conducted another experiment with different concentrations of target. Figure 8 showed the relationship between the reduction of the absorbance and the concentration of the target strand. We found that the higher the concentration of the target was, the lower the signal would be. Reduction in absorbance is directly proportional to the concentration of target, showing that the presence of the target actually reduced the signal. Although it is out of our expectation, it is suggesting that we have accidentally discovered its signal off ability.
We deduced that the cube is very flexible and will easily be closed by the split quadruplex sequences, and we also deduced that the presence of target might just prohibit the cube to close.
The limit of detection (LOD) was calculated to check for the lowest concentration of H3N2 virus DNA this nano-cube could be able to detect. The LOD was calculated as 199.74nM, or 39.9%. H3N2 virus can be detected when concentration of the virus is higher than limit of detection.
3. mRNA Peroxidase Assay Result
Upon achieving positive result from DNA peroxidase assay, we used RNA of the H3N2 virus to conduct an in-vitro test. The steps for this test were similar to DNA peroxidase assay, except DNA of H3N2 virus is replaced by RNA. The results are shown below.
The above graph showed the relationship between the reduction of the absorbance and the concentration of target RNA strand. The result suggested that the reduction in absorbance is directly proportional to the concentration of target RNA strand, which is the same with the result of DNA peroxidase assay. This showed that our nano-cube could not only detect for the DNA strand but also RNA strand. It proves that this nano-cube is suitable to detect virus in which their genetic material are stored as RNA instead of DNA.
The LOD was calculated again to check for the lowest concentration of H3N2 virus RNA this nano-cube could be able to detect. The LOD was calculated as 137.885nM, or 27.6%. H3N2 virus can be detected when concentration of the virus is higher than limit of detection. Obviously, RNA has a much lower limit of detection than DNA.
4. Cloning (Collaborated with Team HKU)
Eight basic parts were submitted to the Registry. These eight basic parts are oligo 1 to oligo 8 and are documented in the table below.
|Type||Name (Part number)||Type||Description||Length (bp)|
|Basic parts||BBa_K2219001||DNA||Oligo 1 for the nano-cube||65|
|Basic parts||BBa_K2219002||DNA||Oligo 2 for the nano-cube||70|
|Basic parts||BBa_K2219003||DNA||Oligo 3 for the nano-cube||52|
|Basic parts||BBa_K2219004||DNA||Oligo 4 for the nano-cube||62|
|Basic parts||BBa_K2219005||DNA||Oligo 5 for the nano-cube||39|
|Basic parts||BBa_K2219006||DNA||Oligo 6 for the nano-cube||23|
|Basic parts||BBa_K2219007||DNA||Oligo 7 for the nano-cube||83|
|Basic parts||BBa_K2219008||DNA||Oligo 8 for the nano-cube||52|
A series of experiments have been conducted to support the evidence that eight oligos we designed were successfully assembled to form a nano-cube. This nano-cube has the diagnostic ability for H3N2 virus by detecting the presence of its mRNA. The reduction in signal of peroxidase assay is directly proportional to the concentration of the mRNA strand.