Difference between revisions of "Team:NCTU Formosa/Composite Part"

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     <p class="content">4. Eco-friendly: Compare with chemical pesticides, Omega-hexatoxin-Hv1a will not remain in soil and water so that it will not pollute the environment and won’t harm the ecosystem. </p>
 
     <p class="content">4. Eco-friendly: Compare with chemical pesticides, Omega-hexatoxin-Hv1a will not remain in soil and water so that it will not pollute the environment and won’t harm the ecosystem. </p>
 
     <p class="content">Altogether, using Hv1a is totally an environmentally friendly way for solving harmful insect problems by using this ion channel inhibitor as a biological pesticide.</p>
 
     <p class="content">Altogether, using Hv1a is totally an environmentally friendly way for solving harmful insect problems by using this ion channel inhibitor as a biological pesticide.</p>
−
     <p class="content"></p>
+
 
−
     <p class="content"></p>
+
     <p class="content-1">Target insect:</p>
 +
    <li class="list">House cricket (<i>Acheta domesticus</i>) </li>
 +
    <li class="list"><i>Musca domestica</i></li>
 +
    <li class="list"><i>Amblyomma americanum</i></li>
 +
    <li class="list"><i>Heliothis virescens</i></li>
 +
 
 +
     <p class="content-1">Experiment</p>
 +
    <p class="content-2">1. Cloning </p>
 +
    <p class="content">After assembling the DNA sequences from the basic parts, we recombined each T7 Promoter+B0034+toxin +linker+6xHistag  gene to pSB1C3 backbones and conducted a PCR experiment to check the size of each part. The DNA sequence length of these parts is around 250-500 bp. In this PCR experiment, the toxin product's size should be near at 450-700 bp. </p>
 +
        <div>
 +
            <img src="a">
 +
        </div>
 +
    <p class="content">proved that we successfully ligated the toxin sequence onto an ideal backbone.</p>
 +
    <p class="content-2">2. Expressing</p>
 +
    <p class="content"><i>E.coli</i>(DE3) express the protein and form the disulfide in the cytoplasm. We sonicated the bacteria and purified the protein by 6xHis-tag behind the toxin using Nickel resin column. </p>
 +
    <p class="content-2">3. Analysis</p>
 +
    <p class="content">We do the Bradford analysis to get the protein concentration. </p>
 +
        <div>
 +
            <img src="a">
 +
        </div>
 +
    <p class="content">Also, we do the UV test and model the degradation rate.</p>
 +
        <div>
 +
            <img src="a">
 +
        </div>
 +
    <p class="content">We did the feeding assay on the Tabacco cutworm (Spodoptera litura) to calculate the LC<sub>50/sub>.</p>
 +
    <p class="content">放蟲抽搐、死亡之影片</p>
 +
    <p class="content-2">4.Modeling</p>
 +
    <p class="content">According to reference, the energy of Ultraviolet will break the disulfide bonds and the toxicity is also decreased. To take the parameter into consideration for our automatic system, we modeled the degradation rate of the protein and modified the program in our device.</p>
 +
        <div>
 +
            <img src="a">放預測降解速率的圖
 +
        </div>
 +
    <p class="content-2">5. Device</p>
 +
    <p class="content">We designed a device that contains a detector, a sprinkler, and an integrated hardware with users by APP through IoT talk. We use an infrared detector to detect the number of the pest and predict what time to spray the farmland. Furthermore, other detectors like temperature, humidity, lamination, pressure of carbon dioxide are also installed in our device. At the same time, the APP that displays all the information about the farmland would contact the users and spray biological pesticides automatically. This device can make farmers control the farmland remotely.</p>
 +
        <div>
 +
            <img src="a">放device的真實圖
 +
        </div>
 +
 
 
     </div>
 
     </div>
  
 
   <div>
 
   <div>
 
     <p class="title">Experience assistance</p>
 
     <p class="title">Experience assistance</p>
−
     <p class="content-1">Distribution kit</p>
+
     <p class="content"></p>
−
     <p class="content-1">NTHU_Taiwan iGEM team</p>
+
     <p class="content"></p>
−
     <p class="content">We provided NTHU_Taiwan iGEM team with BBa_B0012.<br>(NTHU linker)</p>
+
     <p class="content"></p>
−
     <p class="content-1">Mingdao</p>
+
     <p class="content"></p>
−
     <p class="content">We sent the biobricks BBa_K1694002, BBa_K1694010, BBa_K1694035, BBa_K1694044 and BBa_K1694045, which was registry by 2015 NCTU_Formosa, to help them express the outer membrane protein.
+
     <p class="content"></p>
−
<br>(Mingdao linker)</p>
+
     <p class="content"></p>
−
    <p class="content-1">NCKU_Tainan</p>
+
     <p class="content"></p>
−
    <p class="content">This year, it is the first time for NCKU_Tainan to participate in Giant Jamboree. Therefore, we provided them with the Part BBa_K861171, BBa_I712667, BBa_I712015, BBa_R0010, BBa_R0040, BBa_C0040, BBa_C0051, BBa_C0061,  BBa_C0062, BBa_R0065, BBa_K592009 and BBa_R0052 to them.
+
−
<br>(NCKU_Tainan linker)</p>
+
−
     <p class="content-1">Provided larvae by NYMU-Taipei</p>
+
−
     <p class="content">We are thankful to NYMU iGEM team for providing us the eggs of tobacco cutworms. For our insect test, we needed a great number of larvae to do the experiment. Additionally, they introduce the professor Hwang, who majors in agricultural science, and gave us advice in our insect test. We really appreciate the help of NTMU-Taipei.</p>
+
 
   </div>
 
   </div>
  
 
   <div>
 
   <div>
 
     <p class="title">Mentoring a new team</p>
 
     <p class="title">Mentoring a new team</p>
−
   
+
     <p class="content"></p>
−
    <div class="box">
+
     <p class="content"></p>  
−
        <img  src="https://static.igem.org/mediawiki/2016/1/19/Colloboration1.jpg" class="picture">
+
     <p class="content">t</p>
−
    </div>
+
     <p class="content"></p>  
−
   
+
−
     <p class="content-1">National Taichung First Senior High School</p>
+
−
    <li class="list">Learning about iGEM Competition</li>
+
−
    <li class="list">Concept of Synthetic Biology</li>
+
−
    <li class="list">Basic knowledge of Synthetic Biology</li>
+
−
    <li class="list">Basic Mathematical Modelling</li>
+
−
    <li class="list">Laboratory Practice</li>
+
−
    <li class="list">Project Brainstorming</li>
+
−
    <li class="list">Presentation Skill Guidance</li>
+
−
     <p class="content">This summer, fourteen high school students in science class from National Taichung First Senior High School came to National Chiao Tung University to learn about iGEM. In the two-weeks workshop, they have learned a lot of basic concepts of synthetic biology associated with laboratory practices. Furthermore, they were grouped into two teams and assigned to acquire ideas and knowledge from 2015 high school team’s wikis. It not only broadened their horizons but sorted out their thoughts through the presentation. </p>
+
−
 
+
−
     <div class="box">
+
−
        <img  src="https://static.igem.org/mediawiki/2016/7/7c/Colloboration2.jpg" class="picture">
+
−
        <p class="content-image" style="text-align:center;">See <a>Education</a> for the workshop we are planning on.</p>
+
−
    </div>
+
−
   
+
−
     <p class="content">On the last day of the workshop, each group presented their own project, which contained brainstorming, principles, and experimental designs. Their projects were creative, and yet there is still room for the more rigorous thinking. Through a series of compact training, we believe that they will be a competent high school team in the future iGEM competition.</p>
+
−
    <p class="content-1">National Chung Cheng University</p>
+
−
    <li class="list">Experience sharing of founding an iGEM team</li>
+
−
    <li class="list">Teaching Demonstration</li>
+
−
    <li class="list">How do Write Biobrick Sequences</li>
+
−
    <li class="list">Details of Biobrick construction</li>
+
−
    <p class="content">This year, the student leader of National Chung Cheng University came to National Chiao Tung University to learn about the experience of establishing a new team. We exchanged the experience of team operation and academic advice. Besides, she also joined the two-week workshop with students from National Taichung First Senior High School. National Chung Cheng University will establish its first team next year. We are glad to have another companion in Taiwan.</p>
+
−
   
+
−
    <div>
+
−
        <img  src="https://static.igem.org/mediawiki/2016/c/cd/Colloboration3.jpg" class="picture">
+
−
    </div>
+
−
   
+
 
   </div>
 
   </div>
 
</section>
 
</section>

Revision as of 22:03, 29 September 2016

Omega-hexatoxin-Hv1a

T7 Promoter+B0034+Hv1a+linker+6xHistag

Introduction:

By ligating the IPTG induced promoter T7 (BBa_ I712074), strong ribosome binding site (BBa_B0034), hv1a, linker, and the 6xHistag (BBa_ K1223006), we can express Hv1a, the toxin by IPTG induction.

This year we create a revolutionary system that integrates biological pesticides, an automatic detector, a sprinkler, and IoT. We made a database that contains most of the spider toxins and selected the target toxins by programming. Omega-hexatoxin-Hv1a is coded for the venom of a spider, Hadronyche versuta. It is under the control of the strong T7 promoter. A 6xHistag is added for further protein purification.

Mechanism of Hv1a

According to the reference, Omega-hexatoxin-Hv1a has a structure called ICK(inhibitor cysteine knot).[1] This kind of structure contains three disulfide bonds and beta-sheet. With this structure, Hv1a can resist the high temperature, acid base solution and the digest juice of insect gut. Hv1a can bind on insect voltage-gated Calcium channels (CaV1) in the central nervous system, making it paralyze and die eventually.

Features of Hv1a

1. Non-toxic: Omega-hexatoxin-Hv1a is non-toxic to mammals and Hymenoptera (bees). Since the structure of the target ion channel is different, omega-hexatoxin-Hv1a does not harm mammals and bees. So it is safe to use it as a biological pesticide.

2. Biodegradable: Omega-hexatoxin-Hv1a is a polypeptide so it must degrade over time. After degradation, the toxin will become nutrition in the soil.

3. Species-specific: According to reference, Omega-hexatoxin-Hv1a has specificity to Lepidopteran (moths), Dipteran (flies) and Orthopteran (grasshoppers).

4. Eco-friendly: Compare with chemical pesticides, Omega-hexatoxin-Hv1a will not remain in soil and water so that it will not pollute the environment and won’t harm the ecosystem.

Altogether, using Hv1a is totally an environmentally friendly way for solving harmful insect problems by using this ion channel inhibitor as a biological pesticide.

Target insect:

  • House cricket (Acheta domesticus)
  • Musca domestica
  • Amblyomma americanum
  • Heliothis virescens

    • Experiment

    1. Cloning

    After assembling the DNA sequences from the basic parts, we recombined each T7 Promoter+B0034+toxin +linker+6xHistag gene to pSB1C3 backbones and conducted a PCR experiment to check the size of each part. The DNA sequence length of these parts is around 250-500 bp. In this PCR experiment, the toxin product's size should be near at 450-700 bp.

    proved that we successfully ligated the toxin sequence onto an ideal backbone.

    2. Expressing

    E.coli(DE3) express the protein and form the disulfide in the cytoplasm. We sonicated the bacteria and purified the protein by 6xHis-tag behind the toxin using Nickel resin column.

    3. Analysis

    We do the Bradford analysis to get the protein concentration.

    Also, we do the UV test and model the degradation rate.

    We did the feeding assay on the Tabacco cutworm (Spodoptera litura) to calculate the LC50/sub>.

    放蟲抽搐、死亡之影片

    4.Modeling

    According to reference, the energy of Ultraviolet will break the disulfide bonds and the toxicity is also decreased. To take the parameter into consideration for our automatic system, we modeled the degradation rate of the protein and modified the program in our device.

    放預測降解速率的圖

    5. Device

    We designed a device that contains a detector, a sprinkler, and an integrated hardware with users by APP through IoT talk. We use an infrared detector to detect the number of the pest and predict what time to spray the farmland. Furthermore, other detectors like temperature, humidity, lamination, pressure of carbon dioxide are also installed in our device. At the same time, the APP that displays all the information about the farmland would contact the users and spray biological pesticides automatically. This device can make farmers control the farmland remotely.

    放device的真實圖

    Experience assistance

    Mentoring a new team

    t