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− | {{IGEM_TopBar}}
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− | {{Shanghai_high_school}}
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| <html> | | <html> |
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| + | <link rel="stylesheet" type="text/css" |
| + | href="https://2021.igem.org/wiki/index.php?title=Template:Shanghai_high_school/StyleCSS&action=raw&ctype=text/css"/> |
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− | <div class="column full_size judges-will-not-evaluate"> | + | <div class="dr-oral-content"> |
− | <h3>★ ALERT! </h3> | + | <!--navbar--> |
− | <p>This page is used by the judges to evaluate your team for the <a href="https://2021.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2021.igem.org/Judging/Awards"> award listed below</a>. </p> | + | <div class="navbar"> |
− | <p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2021.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
| + | <a class="logo" href="https://2021.igem.org/Team:Shanghai_high_school"> |
− | </div>
| + | <img src="https://static.igem.org/mediawiki/2021/7/7f/T--Shanghai_high_school--logo.png" alt=""> |
| + | </a> |
| | | |
| + | <!--nav--> |
| + | <ul class="list"> |
| + | <!--Team--> |
| + | <li class="item"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Team">Team</a> |
| + | <div class="child-nav"> |
| + | <ul class="child-list"> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Team">Team Members</a> |
| + | </li> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Attributions">Attributions</a> |
| + | </li> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Collaborations">Collaboration</a> |
| + | </li> |
| + | </ul> |
| + | </div> |
| + | </li> |
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− | <div class="clear"></div> | + | <!--Modeling--> |
| + | <li class="item"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Model">Modeling</a> |
| + | </li> |
| | | |
| + | <!--Entrepreneurship--> |
| + | <li class="item"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Entrepreneurship">Entrepreneurship</a> |
| + | </li> |
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− | <div class="column full_size"> | + | <!--Implementation--> |
− | <h1>Engineering Success</h1> | + | <li class="item"> |
− | <h3> Silver Medal Criterion #1</h3>
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Implementation">Implementation</a> |
| + | </li> |
| | | |
− | <p>Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze. | + | <!--Human Practice--> |
− | <br><br> | + | <li class="item"> |
− | Please see the <a href="https://2021.igem.org/Judging/Medals">2021 Medals Page</a> for more information.
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Human_Practices">Human Practice</a> |
− | </p> | + | <div class="child-nav"> |
| + | <ul class="child-list"> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Human_Practices">Integrated Human |
| + | Practice</a> |
| + | </li> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Communication">Communication</a> |
| + | </li> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Fundraising">Fundraising</a> |
| + | </li> |
| + | </ul> |
| + | </div> |
| + | </li> |
| | | |
| + | <!--Parts--> |
| + | <li class="item active"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Parts">Parts</a> |
| + | <div class="child-nav"> |
| + | <ul class="child-list"> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Parts">Parts |
| + | Collection</a> |
| + | </li> |
| + | <li class="child-item active"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Engineering">Engineering</a> |
| + | </li> |
| + | </ul> |
| + | </div> |
| + | </li> |
| | | |
− | <p>If you plan to show engineering success by creating a new Part that has been shown to work as expected, you must document your contribution on the Part's Main Page on the <a href="http://parts.igem.org/Main_Page">Registry</a> for your team to be eligible for this criteria.</p> | + | <!--Project--> |
| + | <li class="item"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Description">Project</a> |
| + | <div class="child-nav"> |
| + | <ul class="child-list"> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Description">Description</a> |
| + | </li> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Experiments">Experiments</a> |
| + | </li> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Results">Results</a> |
| + | </li> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Proof_Of_Concept">Proof of Concept</a> |
| + | </li> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Notebook">Notebook</a> |
| + | </li> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school/Safety">Safety</a> |
| + | </li> |
| + | </ul> |
| + | </div> |
| + | </li> |
| | | |
− | </div> | + | <!--Home--> |
| + | <li class="item"> |
| + | <a href="https://2021.igem.org/Team:Shanghai_high_school">Home</a> |
| + | </li> |
| + | </ul> |
| + | </div> |
| | | |
| + | <!--图片--> |
| + | <div class="page-header"> |
| + | <img src="https://static.igem.org/mediawiki/2021/f/fd/T--Shanghai_high_school--bg_1.png" alt=""> |
| + | <h1>Engineering</h1> |
| + | </div> |
| | | |
| + | <!--内容--> |
| + | <div class="content-small"> |
| + | <section class="article p-t-54 p-b-54"> |
| + | <section> |
| + | <h1 class="title">Introduction </h1> |
| + | |
| + | <p>Rotavirus (RV) is the main viral pathogen that causes severe acute diarrhea in infants and young |
| + | children. Almost all children under five weeks of age have been infected with the virus, causing nearly |
| + | 130,000 deaths worldwide each year. Social conditions in developing countries have led to reduced |
| + | effectiveness of oral rehydration solutions and vaccines, as well as a lack of approved antiviral drugs, |
| + | making rotavirus infection a global health problem. RV structural protein vp7, on the outermost layer of |
| + | virus particles, is the first choice for the development of genetic engineering vaccines. We are trying |
| + | to develop a new oral vaccine for hand, foot and mouth disease due to its advertisement for infants and |
| + | young children.</p> |
| + | |
| + | <p>The B subunit LTB in the heat-labile enterotoxin (LT) of Escherichia coli heat-labile enterotoxin (LT) |
| + | has strong immunogenicity and adjuvant activity, and will not cause harm to the human body. LTB and a |
| + | variety of non-related proteins and their non-protein antigens can increase the mucosal IgA and humoral |
| + | immune IgG response levels of the antigen through different immunization pathways. Currently, there are |
| + | three main types of vaccines, including inactivated vaccines/attenuated vaccines, mRNA vaccines/DNA |
| + | vaccines, and neutralizing antibody/non-neutralizing antibody vaccines. Human vaccination methods |
| + | include injection (hepatitis B vaccine, BCG vaccine, flu vaccine, etc.) and oral administration |
| + | (poliomyelitis, cholera vaccine, and rotavirus vaccine). </p> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">Design </h1> |
| + | |
| + | <p>Bacillus subtilis is an important probiotic in human intestinal tract and is safe and has no endotoxin. |
| + | It is an ideal oral live vaccine expression vector. Recombinant foreign protein does not need to be |
| + | purified and can be taken directly with the bacteria. We constructed a <i>Bacillus subtilis</i> |
| + | expression system to express rotavirus VP7-LTB fusion protein. As a mucosal immune adjuvant, LTB |
| + | enhances the antiviral ability of vp7. This oral vaccine has broad clinical application prospects. </p> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">Build</h1> |
| + | |
| + | <p>Four recombinant plasmids were constructed. They are pET-28a(+)-vp7, pET-28a(+)-vp7-LTB (Figure 1), |
| + | PHT43-HIS-VP7, and PHT43-HIS-VP7-LTB. The constructs of pET-28a expression vector and PHT43-HIS are used |
| + | for recombinant protein production in <i>E. coli</i> and <i>B. subtilis</i>, respectively. The vp7 and |
| + | LTB genes were obtained by gene synthesis according to the deposited sequence from GenBank (AY165008.1 |
| + | and AF242418.1, respectively. The plasmids were constructed by homologous recombination. All the |
| + | constructs are confirmed by DNA sequencing.</p> |
| + | |
| + | <div class="img-container"> |
| + | <img src="https://static.igem.org/mediawiki/2021/e/e5/T--Shanghai_high_school--img_engineering_1.jpg" alt="" style="width: 70%"> |
| + | <span class="figure">Figure 1. Schematic map of plasmids.</span> |
| + | </div> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">Test</h1> |
| + | |
| + | <p>SDS-PAGE shows the protein expression in <i>E. coli</i> (Figure 2). VP7 and VP7-LTB protein appears in |
| + | the precipitates which suggests that the proteins from inclusion body. IPTG may be a factor, 1nm is a |
| + | suitable concentration. LTB stimulates the expression of VP7.</p> |
| + | |
| + | <div class="img-container"> |
| + | <img src="https://static.igem.org/mediawiki/2021/5/59/T--Shanghai_high_school--img_engineering_2.jpg" alt="" style="width: 80%"> |
| + | <span class="figure">Figure 1. Schematic map of plasmids.</span> |
| + | </div> |
| + | |
| + | <p>With induction by IPTG, <i>B. subtilis</i> WB800n did not find the target protein from SDS-PAGE (Figure |
| + | 3).</p> |
| + | |
| + | <div class="img-container"> |
| + | <img src="https://static.igem.org/mediawiki/2021/3/31/T--Shanghai_high_school--img_engineering_3.jpg" alt="" style="width: 80%"> |
| + | <span class="figure">Figure 3. SDS-PAGE analysis of expression of VP7 and VP7-LTB in <i>B. subtilis</i> WB800n.</span> |
| + | </div> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">Learn</h1> |
| + | |
| + | <p>VP7 and VP7-LTB were successfully expression in <i>E. coli</i> predominantly as inclusion bodies. The |
| + | protein expression in <i>B. subtilis</i> was not successful as indicated by SDS-PAGE and Western blot. |
| + | </p> |
| + | </section> |
| + | </section> |
| + | </div> |
| + | |
| + | <!--Footer--> |
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| + | <div class="content"> |
| + | <hr> |
| + | <span>Contact Email: 1649407718@qq.com; 2796437537@qq.com</span> |
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