Difference between revisions of "Team:HK SKHLPSS"

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         All in all, these two gel results suggested that our nano-cube should be formed as per our design.
 
         All in all, these two gel results suggested that our nano-cube should be formed as per our design.
 
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       <h2>2. DNA Peroxidase Assay Result</h2>
 
       <h2>2. DNA Peroxidase Assay Result</h2>
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         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.
 
         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.
 
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         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.
 
         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.
 
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       <h2>4. Cloning (Collaborated with Team HKU)</h2>
 
       <h2>4. Cloning (Collaborated with Team HKU)</h2>

Revision as of 06:34, 1 November 2017

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Results

1. Gel electrophoresis (Collaborated with Team HKU)

Fig. 5. Result of the first gel. The last lane clearly showed that all 8 oligos are binded together.

With the help of HKU team, we are able to get the gel electrophoresis to analyze whether our design was actually assembled. As seen in the first gel result. All 8 oligos come together as the shift observed in last lane.

Fig. 6. Result of second gel. The second gel further proves that our nano-cube is formed according to our conceptual design.

To further proof 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 binded 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 binded with the nano-cube.

All in all, these two gel results suggested that our nano-cube should be formed as per our design. 

     Template:Clear

2. DNA Peroxidase Assay Result

Fig. 7. The result of DNA peroxidase assay. It showed that the presence of target together with the nano-cube would lead to the reduction of absorbance.
Fig. 8. The result of DNA peroxidase assay in different concentration of target. The above graph suggested that reduction of the absorbance is directly proportional to the concentration

These two graphs are our results. In the first graph, it 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. Then, we conducted another experiment with different concentrations of target. The second graph shows 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 was. 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.

Fig. 9. Deduction of the nano-cube in the presence of target. It is deduced that the presence of target might prohibit the cube to close.

We deduced the cube is very flexible and will easily be closed by the split quadruplex sequences and we deduced that the presence of target might just prohibit the cube to close.

Fig 10. Limit of Detection Formula. The LOD is calculated according to above formula.

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. 

     Template:Clear

3. mRNA Peroxidase Assay Result

Upon successful of 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.

Fig. 11. The result of RNA peroxidase assay. It showed that the presence of mRNA of the target together with the nano-cube would also lead to the reduction of absorbance and is also in linear relationship.

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. 

     Template:Clear

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 below table.

<tbody> </tbody>
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

5. Conclusion

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 intensity of peroxidase assay is directly proportional to the concentration of the mRNA strand.