Difference between revisions of "Team:SCAU-China/HP/Silver"

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<li><a href="https://2016.igem.org/Team:SCAU-China/Time_shaft">Time shaft</a></li>
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<li><a href=" https://2016.igem.org/Team:SCAU-China/Experiments"> Experiments  </a></li>
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<li><a href=" https://2016.igem.org/Team:SCAU-China/Notebook">Notebook</a></li>
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      <li><a href="#Parts" class="on">Parts Table</a></li>
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<li><a href="https://2016.igem.org/Team:SCAU-China/Gallery">Gallery</a></li>
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      <li><a href="#Origin">Origin and function</a></li>
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<li><a href="https://2016.igem.org/Team:SCAU-China/Protocol">Protocol</a></li>
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      <li><a href="#Semi">Semi-quantitative RT-PCR analysis</a></li>
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  <li><a href="#Phenotype">Phenotype of transgenic rice</a></li>
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  <li><a href="#Pigment">Pigment analysis</a></li>
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<li><a href="https://2016.igem.org/Team:SCAU-China/Safety">Safety</a></li>
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  <li><a href="#Investigation">Investigation on T2 generation astaxanthin-produing rice</a></li>
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<li><a href="   https://2016.igem.org/Team:SCAU-China/HP/Silver">Silver</a></li>
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<li><a href=" https://2016.igem.org/Team:SCAU-China/HP/Gold">Gold </a></li>
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<li><a href="https://2016.igem.org/Team:SCAU-China/Integrated_Practices">Integrated</a></li>
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<li><a href=" https://2016.igem.org/Team:SCAU-China/Engagement">Engagement</a></li>
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<li><a href=" https://2016.igem.org/Team:SCAU-China/Entrepreneurship">Entrepreneurship</a></li>
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<div class="h1_font_size">Silver</div>
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<div class="p_font_size">Our human practice work involved several questions relating to biosafety,social justice and public acceptance.Our astarice is actually a genetically modified organism,so our work focus on people’s concern about GMO.</div>
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<div class="h2_font_size">1. Improve ourselves</div>
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<div class="p_font_size">First of all,we should make sure that we ourselves have correct understanding on this question.For this reason,we took part in lectures about transgenic.</div>
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<div class="row" style="margin-top:20px">
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<img alt="image" class="img-responsive col s11" src="https://static.igem.org/mediawiki/2016/7/74/T--SCAU-China--experiement1.jpg">
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</div>
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<div class="col s6">
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<div class="p_font_size">The first lecture was given by Kou Jianping ,who is director of the Department of “genetically modified organisms safety management and Intellectual Property Office” from “Department of Science, Technology and Education” in MOA(Minstry of Agriculture) of China,and Lin Min,who is director of Institute of Biotechnology of CAAS(Chinese Academy of Agricultural Sciences).In this lecture，we got to know that transgenic foods,that are saling in market are absolutely safe.For they were tested by associated departments and labs for many years in scientific way.And up to now，there're only a few kinds of transgenic products have gotten permission into Chinese market. After lecture,we communicated with this scientists about new technologies on transgenic research.</div>
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<div class="col s6">
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<div class="p_font_size">Apart from that,we've also took part in a talk given by Wu Yongning,who is the director of “Key Laboratory of Food Safety Risk Assessment,Ministry of Health”,which belongs to China National Center forFood Safety Risk Assessment (CFSA).In this talk,we obtained a lot of informations in analysing GMO.</div>
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<div class="p_font_size">As we are working to create a new kind of crop,we think we should also know more about agriculture,so we joined in The Sixth Asian Conference on Precision  Agriculture as voulnteers.</div>
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<div class="col s6" align="center">
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<img alt="image" class="img-responsive col s11" src="https://static.igem.org/mediawiki/2016/7/74/T--SCAU-China--experiement1.jpg">
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      <div class="text">The parts we submit are listed at the table below.<br><br>
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<script src="https://2016.igem.org/Team:SCAU-China/jsfile?action=raw&ctype=text/javascript"></script> <!--mater-->
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    <td width="119">Part name</td>
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    <td width="273">Description</td>
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    <td width="65">Length</td>
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  </tr>
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    <td><p ><a href="http://parts.igem.org/Part:BBa_K1959000" >BBa_K1959000</a></p></td>
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    <td><p >PSY cds codon optimized for O.sativa.</p></td>
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    <td><p >1193 bp</p></td>
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  </tr>
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  <tr>
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    <td><p ><a href="http://parts.igem.org/Part:BBa_K1959001" >BBa_K1959001</a></p></td>
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    <td><p >Ctr&#8544; cds codon optimized for O.sativa.</p></td>
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    <td><p >1650 bp</p></td>
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  </tr>
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    <td><p ><a href="http://parts.igem.org/Part:BBa_K1959002" >BBa_K1959002</a></p></td>
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    <td><p >BHY cds codon optimized for O.sativa.</p></td>
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    <td><p >1053 bp</p></td>
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  </tr>
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    <td><p ><a href="http://parts.igem.org/Part:BBa_K1959003" >BBa_K1959003</a></p></td>
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    <td><p >BKT cds codon optimized for O.sativa.</p></td>
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    <td><p >1158 bp</p></td>
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  </tr>
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</table>
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      <a id="Origin"></a>
 
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    <li><a href="#Parts">Parts Table</a></li>
 
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      <li><a href="#Origin" class="on">Origin and function</a></li>
 
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      <li><a href="#Semi">Semi-quantitative RT-PCR analysis</a></li>
 
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  <li><a href="#Phenotype">Phenotype of transgenic rice</a></li>
 
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  <li><a href="#Pigment">Pigment analysis</a></li>
 
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  <li><a href="#Investigation">Investigation on T2 generation astaxanthin-produing rice</a></li>
 
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      </ul>
 
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      <div class="text">
 
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  (1) K195900<br><br>
 
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  This part is the coding sequence of phytoene synthase (PSY) from Zea mays. Codon optimized for expression in Orazy sativa. PSY is part of beta-carotene biosynthesis pathway and it catalyzes the conversion of gerarylgeranyl diphosphate to phytoene.
 
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      <img src="https://static.igem.org/mediawiki/2016/4/48/T--SCAU-China--part1.png">
 
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  Reaction catalyzed by PSY<br><br>
 
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  2 geranylgeranyl diphosphate → 15-cis-phytoene + diphosphate.<br><br>
 
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  (2) K1959001<br><br>
 
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  This part is the phytoene desaturase (Crt I) from Erwinia uredovora fused with Pea transit peptide. It is codon optimized for Orazy sativa. Bacterial phytoene desaturase catalyzes conversion of phytoene to lyscoene.
 
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</div>
 
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  <div class="img">
 
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      <img src="https://static.igem.org/mediawiki/2016/f/f8/T--SCAU-China--part2.png">
 
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      </div>
 
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<div class="text">
 
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  Reaction catalyzed by Crt I<br><br>
 
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  15-cis-phytoene + 4 Acceptors → all-trans-lycopene + 4 Reduced acceptors<br><br>
 
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  （3）K1959002<br><br>
 
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This part is the beta-carotene hydroxylase (BHY) coding sequence from Haematococcus pluvialis. The Pea transit peptide is functionally fused with BHY in order to help BHY polypeptide folding correctly. BHY sequence is codon optimized for expression in Orazy sativa. The BHY is part of astaxanthin biosynthesis pathway and it catalyzes conversion of beta-carotene to zeaxanthin.
 
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</div>
 
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  <div class="img">
 
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      <img src="https://static.igem.org/mediawiki/2016/2/20/T--SCAU-China--part3.png">
 
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<div class="text">
 
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  Reaction catalyzed by BHY<br><br>
 
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  Beta-carotene + 2NADH + Oxygen → Zeaxanthin + 2NAD+ + 2H2O<br><br>
 
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  (4)K1959003<br><br>
 
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This part containg Pea transit peptide and coding sequence of beta-carotene ketolase (BKT). BKT catalyzes zeaxanthin to astaxanthin. Codon optimization has been made for expression in Orazy sativa.
 
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Reaction catalyzed by BKT
 
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</div>
 
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  <div class="img">
 
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      <img src="https://static.igem.org/mediawiki/2016/6/63/T--SCAU-China--part4.png">
 
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      </div>
 
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<div class="text">
 
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  Zeaxanthin + 2 Oxygen → Astaxanthin + 2H2O
 
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</div>
 
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    </div>
 
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    <div class="vecter">
 
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      <a id="Semi"></a>
 
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      <li><a href="#Parts">Parts Table</a></li>
 
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      <li><a href="#Origin">Origin and function</a></li>
 
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      <li><a href="#Semi" class="on">Semi-quantitative RT-PCR analysis</a></li>
 
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  <li><a href="#Phenotype">Phenotype of transgenic rice</a></li>
 
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  <li><a href="#Pigment">Pigment analysis</a></li>
 
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  <li><a href="#Investigation">Investigation on T2 generation astaxanthin-produing rice</a></li>
 
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      </ul>
 
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      <div class="text">Transcription levels of the four transgenes (PSY, Crt I, BHY, BKT) are analyzed by performing semi-quantitative RT-PCR, Actin is served as internal control. Total RNA is extracted from astaxanthin-containing seeds and amplified using specific primers. Expected band representing the correspond genes can be detected in transformants while no band is observed in wild-type. <br><br>
 
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  The RT-PCR result demonstrates that all of four transgenes is capable to transcript in rice endosperm. To a certain extent, the result also indicates that the astaxanthin biosynthesis pathway is worked in the rice endosperm.
 
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</div> 
 
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  <div class="img">
 
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      <img src="https://static.igem.org/mediawiki/2016/d/dc/T--SCAU-China--part5.png">
 
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        <div class="text">Fig.N    RT-PCR analysis of expression of CrtI, PSY, BKY,BHT genes intransformants, Actin serves as internal control. CK+, positive control (PCR product amplified from plasmid pYLTAC380MF-BBPC using specific primers ). <br><br>
 
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  WT, negetive control (RT-PCR product of HG1 total RNA).
 
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      <a id="Phenotype"></a>
 
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      <li><a href="#Parts">Parts Table</a></li>
 
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      <li><a href="#Origin">Origin and function</a></li>
 
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      <li><a href="#Semi">Semi-quantitative RT-PCR analysis</a></li>
 
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  <li><a href="#Phenotype" class="on">Phenotype of transgenic rice</a></li>
 
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  <li><a href="#Pigment">Pigment analysis</a></li>
 
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  <li><a href="#Investigation">Investigation on T2 generation astaxanthin-produing rice</a></li>
 
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      </ul>
 
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      <div class="text">Polished transgenic seeds present in a orange color, indicating certain astaxanthin content. Wild-type rice without astaxanthin accumulation remains colorless. Inside situation of the endosperm is shown at the cross-section photo. The entire endosperm is stained in orange, representing the homogeneous distribution of astaxanthin.
 
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</div> 
 
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  <div class="img">
 
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      <p><img src="https://static.igem.org/mediawiki/2016/4/4b/T--SCAU-China--part6.png"></p>
 
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  <p><img src="https://static.igem.org/mediawiki/2016/a/a4/T--SCAU-China--part7.png"></p>
 
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      </div>
 
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  <div class="text">Fig.n  The top and cross-sectional view of phenotype of Huguang1 (O.sativa.spp.indica) wild-type and transgenic rice.
 
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      <a id="Pigment"></a>
 
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      <li><a href="#Parts">Parts Table</a></li>
 
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      <li><a href="#Origin">Origin and function</a></li>
 
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      <li><a href="#Semi">Semi-quantitative RT-PCR analysis</a></li>
 
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  <li><a href="#Phenotype">Phenotype of transgenic rice</a></li>
 
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  <li><a href="#Pigment" class="on">Pigment analysis</a></li>
 
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  <li><a href="#Investigation">Investigation on T2 generation astaxanthin-produing rice</a></li>
 
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      </ul>
 
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      <div class="text">The T1 generation seeds of wild-type and transgenic lines are subjected to astaxanthin extraction and high performance liquid chromatography (HPLC) to analyze the pigment composition and astaxanthin content. Astaxanthin is indentified on the basis of retention times relative to standard compound. <br><br>
 
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  Content is determined by integrating peak areas and converted to concentration. According to the retention time of standard astaxanthin compound, astaxanthin production in rice endosperm can be confirmed. <br><br>
 
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  The HPLC also result shown that astaxanthin is the predominant carotenoid, while some new compounds are synthesized in conjunction with astaxanthin production. These compounds may be the intermediate products during astaxanthin biosynthesis. Further investigations are needed to identify these new compounds. Astaxanthin content in 1g of polished rice in both transgenic and wild-type rice are shown at Table.2 . <br><br>
 
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  Table.2 Astaxanthin content in wild-type and transgenic rice<br><br>
 
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  <table width="285" height="127" border="1" align="center">
 
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  <tr>
 
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    <td width="124" height="48" align="center" valign="middle">HG-WT </td>
 
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    <td width="145" align="center" valign="middle">HG-380MF-BBPC</td>
 
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  </tr>
 
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  <tr>
 
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    <td align="center" valign="middle"><p >0ug/g</p></td>
 
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    <td align="center" valign="middle"><p >7.91 ug/g</p></td>
 
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  </tr>
 
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</table>
 
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      <a id="Investigation"></a>
 
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      <li><a href="#Parts">Parts Table</a></li>
 
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      <li><a href="#Origin">Origin and function</a></li>
 
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      <li><a href="#Semi">Semi-quantitative RT-PCR analysis</a></li>
 
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  <li><a href="#Phenotype">Phenotype of transgenic rice</a></li>
 
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  <li><a href="#Pigment">Pigment analysis</a></li>
 
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  <li><a href="#Investigation" class="on">Investigation on T2 generation astaxanthin-produing rice</a></li>
 
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      </ul>
 
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      <div class="text">The T2 generation (the third generation ) seeds were harvested at the end of September this year and we performed quantitative RT-PCR as well as HPLC analysis subsequently. Several transgenic lines were chosen for investigation. <br><br>
 
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  As the results shown in the diagram, astaxanthin content varies from lines, some lines remain stable astaxanthin production while some synthesize in a low level. Supported by the quantitative RT-PCR analysis, the low content of astaxanthin in the endosperm of transgenic lines are associated with poor transcriptional activity of one of genes (BHY, which no obvious transcriptional activity is detected in four lines). The investigation demonstrates that astaxanthin biosynthesis accomplishes only under the well cooperation of the four genes, repression occurred on just one gene will badly diminish the astaxanthin content. Reasons why BHY gene is inactive in some T2 lines remain unknown and further research is needed to figure out the origins of this phenomenon. 
 
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      <img src="https://static.igem.org/mediawiki/2016/0/04/T--SCAU-China--part8.png">
 
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  <div class="text">Fig N    Relative expression levels of astaxanthin biosynthetic genes and astaxanthin content in seeds of wild-type and T2 generation of transformants.  <br><br>
 
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  The levels of expression are normalized to actin transcript level which is set as 1. <br><br>
 
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  The data represent average values from the analysis of three different plants. <br><br>
 
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  Error bars indicate ± SEM. dwt, dry weight.
 
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Revision as of 11:52, 14 October 2016

SCAU

Silver
Our human practice work involved several questions relating to biosafety,social justice and public acceptance.Our astarice is actually a genetically modified organism,so our work focus on people’s concern about GMO.
1. Improve ourselves
First of all,we should make sure that we ourselves have correct understanding on this question.For this reason,we took part in lectures about transgenic.
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The first lecture was given by Kou Jianping ,who is director of the Department of “genetically modified organisms safety management and Intellectual Property Office” from “Department of Science, Technology and Education” in MOA(Minstry of Agriculture) of China,and Lin Min,who is director of Institute of Biotechnology of CAAS(Chinese Academy of Agricultural Sciences).In this lecture，we got to know that transgenic foods,that are saling in market are absolutely safe.For they were tested by associated departments and labs for many years in scientific way.And up to now，there're only a few kinds of transgenic products have gotten permission into Chinese market. After lecture,we communicated with this scientists about new technologies on transgenic research.
Apart from that,we've also took part in a talk given by Wu Yongning,who is the director of “Key Laboratory of Food Safety Risk Assessment,Ministry of Health”,which belongs to China National Center forFood Safety Risk Assessment (CFSA).In this talk,we obtained a lot of informations in analysing GMO.
As we are working to create a new kind of crop,we think we should also know more about agriculture,so we joined in The Sixth Asian Conference on Precision Agriculture as voulnteers.
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