Difference between revisions of "Team:HK SKHLPSS"

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      <h2> Welcome to iGEM 2017! </h2>
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      <p>Your team has been approved and you are ready to start the iGEM season! </p>
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    <h1>Results</h1>
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   <h1>  A Self-Assembled DNA Nano-cube for the Diagnosis of H3N2 Influenza  </h1>
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  <h2>Abstract</h2>
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      <h2>1. Gel electrophoresis (Collaborated with Team HKU)</h2>
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      [[File: LPResults_1.jpg |thumbnail| left | 500px| Fig. 5. Result of the first gel. The last lane clearly showed that all 8 oligos are binded together.]]
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      [[File: LPResults_2.jpg |thumbnail| left | 500px| Fig. 6. Result of second gel. The second gel further proves that our nano-cube is formed according to our conceptual design.| clear]]
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    DNA is always used a genetic material for information transfer over generations. Its programmability also allows the application in fabricating various objects for diagnostics and therapeutics as the field of DNA nanotechnology. This year, we used DNA to fabricate a three-dimensional cube that is responsive to the presence of H3N2 influenza mRNA biomarker. We used DNA and RNA oligos of the same sequence of the target for detection. From the result, we found that the DNA nano-cube specifically responses to the presence of target leading to the opening of the lid. This results in the disassemble of the quadruplex formation and reduction in hemin-mediated peroxidase activity. We proved that a three-dimensional nanodevice can be used for quick diagnosis within 30 minutes and it is applicable for the detection of different biomarkers and we wish to largely produce this diagnostic device with engineered bacteria.
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        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.
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  The biodiversity of organism is originated from the information carried on DNA that it is only constructed by the fundamental Watson-Crick base pairing rules governing two basic combination of base pairs. The programmability of DNA allows the use of it as LEGO bricks to build different objects in both two-dimensional and three-dimensional manner.
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        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.
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  In addition to showing the possibility of programming DNA into different shapes, the field of DNA nanotechnology was developed to use it for medical purposes. Under the presence of target nucleic acids, conformational change of a molecular beacon that is also consisted of DNA will occur through a simple strand displacement process. Apart from nucleic acids target, presence of metal ions is also detectable by DNA molecular beacon as it affects the stability of DNA. To detect metal ion, G-quadruplex as a four-stranded nucleic acid structure is well known that the formation of it is highly depending on cation. The formation of quadruplex is able to stabilize the molecular structure of hemin and enhance its’ peroxidase activity that leads to the result of quantitative fluorescence or colorimetric signal.
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        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.
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  This year, the ultimate goal of our iGEM team is trying to establish a metal ion responsive molecular beacon in E. coli as a general platform for detecting environmental contamination using bacteria. In order to achieve this progressively, we will first perform the experiment in vitro using molecular beacon that consists of short oligos as a prove of concept. Then we will try to express the system in E. coli and conduct the same experiment using laboratory prepared sample and on-site collected sample for the detection of contamination.
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         All in all, these two gel results suggested that our nano-cube should be formed as per our design.
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    <h3>Before you start: </h3>
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    <p> Please read the following pages:</p>
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    <ul>
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       <li> <a href="https://2017.igem.org/Competition">Competition Hub</a> </li>
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      <li> <a href="https://2017.igem.org/Competition/Deliverables/Wiki">Wiki Requirements page</a></li>
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      <li> <a href="https://2017.igem.org/Resources/Template_Documentation">Template documentation</a></li>
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    </ul>
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  </div>
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  <div class="col-md-6">
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    <div class="highlight">
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       <h3> Styling your wiki </h3>
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       <p>You may style this page as you like or you can simply leave the style as it is. You can easily keep the styling and edit the content of these default wiki pages with your project information and completely fulfill the requirement to document your
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         project.
 
       </p>
 
       </p>
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      <p>While you may not win Best Wiki with this styling, your team is still eligible for all other awards. This default wiki meets the requirements, it improves navigability and ease of use for visitors, and you should not feel it is necessary to style
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      beyond what has been provided.</p>
 
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      <h2>2. DNA Peroxidase Assay Result</h2>
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      [[File: LPResults_3.png |thumbnail| centre | 600px| 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. ]]
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<hr / >
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      [[File: LPResults_4.png |thumbnail| centre | 600px| 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 ]]
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        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.
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    <h3> Wiki template information </h3>
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      [[File: LPResults_5.jpg |thumbnail|centre | 900px| 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.]]
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    <p>We have created these wiki template pages to help you get started and to help you think about how your team will be evaluated. You can find a list of all the pages tied to awards here at the <a href="https://2017.igem.org/Judging/Pages_for_Awards">Pages for awards</a>      link. You must edit these pages to be evaluated for medals and awards, but ultimately the design, layout, style and all other elements of your team wiki is up to you!</p>
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        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.
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       [[File: LPResults_6.jpg |thumbnail| left | 300px| Fig 10. Limit of Detection Formula. The LOD is calculated according to above formula. ]]
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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.
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      </p>
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      <h3> Editing your wiki </h3>
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       <p>On this page you can document your project, introduce your team members, document your progress and share your iGEM experience with the rest of the world! </p>
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       <p> <a href="https://2017.igem.org/wiki/index.php?title=Team:Example&action=edit"> </a>Use WikiTools - Edit in the black menu bar to edit this page</p>
 
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       <h2 style = "text-align:left">3. mRNA Peroxidase Assay Result</h2>
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       <h3>Tips</h3>
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       <p>This wiki will be your team’s first interaction with the rest of the world, so here are a few tips to help you get started: </p>
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        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.
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       <ul>
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      </p>
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        <li>State your accomplishments! Tell people what you have achieved from the start. </li>
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       [[File: LPResults_7.jpg |thumbnail|left | 600px| 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. ]]
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         <li>Be clear about what you are doing and how you plan to do this.</li>
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        <li>You have a global audience! Consider the different backgrounds that your users come from.</li>
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      <p>
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        <li>Make sure information is easy to find; nothing should be more than 3 clicks away. </li>
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         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.
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        <li>Avoid using very small fonts and low contrast colors; information should be easy to read. </li>
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      </p>
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         <li>Start documenting your project as early as possible; don’t leave anything to the last minute before the Wiki Freeze. For a complete list of deadlines visit the <a href="https://2017.igem.org/Calendar">iGEM 2017 calendar</a> </li>
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      <p>
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        <li>Have lots of fun! </li>
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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.
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      <h2>4. Cloning (Collaborated with Team HKU)</h2>
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        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.
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      <h3>Inspiration</h3>
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      <p> You can also view other team wikis for inspiration! Here are some examples:</p>
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            <th style="text-align: center" width="25%"> Type</th>
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      <ul>
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            <th style="text-align: center">Name (Part number)</th>
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        <li> <a href="https://2014.igem.org/Team:SDU-Denmark/"> 2014 SDU Denmark </a> </li>
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            <th style="text-align: center" width="15%"> Type </th>
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        <li> <a href="https://2014.igem.org/Team:Aalto-Helsinki">2014 Aalto-Helsinki</a> </li>
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            <th style="text-align: center" width="15%">Description</th>
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        <li> <a href="https://2014.igem.org/Team:LMU-Munich">2014 LMU-Munich</a> </li>
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            <th style="text-align: center" width="15%">Length (bp)</th>
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        <li> <a href="https://2014.igem.org/Team:Michigan"> 2014 Michigan</a></li>
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          </tr>
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        <li> <a href="https://2014.igem.org/Team:ITESM-Guadalajara">2014 ITESM-Guadalajara </a></li>
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          <tr style="font-size: 0.8em">
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        <li> <a href="https://2014.igem.org/Team:SCU-China"> 2014 SCU-China </a></li>
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            <td style="text-align: center">Basic parts</td>
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       </ul>
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            <td>BBa_K2219001</td>
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            <td style="text-align: center">DNA</td>
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            <td>Oligo 1 for the nano-cube</td>
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            <td style="text-align: center">65</td>
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          </tr>
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          <tr style="font-size: 0.8em">
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            <td style="text-align: center">Basic parts</td>
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            <td>BBa_K2219002</td>
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            <td style="text-align: center">DNA</td>
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            <td>Oligo 2 for the nano-cube</td>
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            <td style="text-align: center">70</td>
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          </tr>
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          <tr style="font-size: 0.8em">
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            <td style="text-align: center">Basic parts</td>
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            <td>BBa_K2219003</td>
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            <td style="text-align: center">DNA</td>
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            <td>Oligo 3 for the nano-cube</td>
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            <td style="text-align: center">52</td>
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          </tr>
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          <tr style="font-size: 0.8em">
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            <td style="text-align: center">Basic parts</td>
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            <td>BBa_K2219004</td>
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            <td style="text-align: center">DNA</td>
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            <td>Oligo 4 for the nano-cube</td>
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            <td style="text-align: center">62</td>
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          </tr>
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          <tr style="font-size: 0.8em">
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            <td style="text-align: center">Basic parts</td>
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            <td>BBa_K2219005</td>
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            <td style="text-align: center">DNA</td>
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            <td>Oligo 5 for the nano-cube</td>
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            <td style="text-align: center">39</td>
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          </tr>
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          <tr style="font-size: 0.8em">
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            <td style="text-align: center">Basic parts</td>
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            <td>BBa_K2219006</td>
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            <td style="text-align: center">DNA</td>
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            <td>Oligo 6 for the nano-cube</td>
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            <td style="text-align: center">23</td>
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          </tr>
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          <tr style="font-size: 0.8em">
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            <td style="text-align: center">Basic parts</td>
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            <td>BBa_K2219007</td>
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            <td style="text-align: center">DNA</td>
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            <td>Oligo 7 for the nano-cube</td>
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            <td style="text-align: center">83</td>
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          </tr>
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          <tr style="font-size: 0.8em">
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            <td style="text-align: center">Basic parts</td>
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            <td>BBa_K2219008</td>
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            <td style="text-align: center">DNA</td>
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            <td>Oligo 8 for the nano-cube</td>
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            <td style="text-align: center">52</td>
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          </tr>
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        </tbody>
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       </table>
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       <h2>5. Conclusion</h2>
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       <h3> Uploading pictures and files </h3>
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        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.
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       <p> You can upload your pictures and files to the iGEM 2017 server. Remember to keep all your pictures and files within your team's namespace or at least include your team's name in the file name. <br /> When you upload, set the "Destination Filename"
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        to
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        <br><code>T--YourOfficialTeamName--NameOfFile.jpg</code>. (If you don't do this, someone else might upload a different file with the same "Destination Filename", and your file would be erased!)<br><br>
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        <a href="https://2017.igem.org/Special:Upload">
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          UPLOAD FILES
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        </a>
 
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Revision as of 06:48, 1 November 2017

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JUDGING FORM

A Self-Assembled DNA Nano-cube for the Diagnosis of H3N2 Influenza

Abstract

DNA is always used a genetic material for information transfer over generations. Its programmability also allows the application in fabricating various objects for diagnostics and therapeutics as the field of DNA nanotechnology. This year, we used DNA to fabricate a three-dimensional cube that is responsive to the presence of H3N2 influenza mRNA biomarker. We used DNA and RNA oligos of the same sequence of the target for detection. From the result, we found that the DNA nano-cube specifically responses to the presence of target leading to the opening of the lid. This results in the disassemble of the quadruplex formation and reduction in hemin-mediated peroxidase activity. We proved that a three-dimensional nanodevice can be used for quick diagnosis within 30 minutes and it is applicable for the detection of different biomarkers and we wish to largely produce this diagnostic device with engineered bacteria.