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

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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="   https://2016.igem.org/Team:SCAU-China/HP/Silver">Silver</a></li>
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<a class="right brand-logo dropdown-button" data-beloworigin="true" data-constrainwidth="false" style="cursor:pointer"  data-activates="dropdown7">HUMAN PRACTICES</a>
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<div class="h1_font_size">Gold</div>
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<div class="p_font_size">Our human practice work involved several questions relating to bio-safety,social justice and public acceptance.Our astarice is actually a genetically modified organism,so our work focused on people’s concern about GMO.</div>
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<div class="h2_font_size">1. Investigation</div>
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<img alt="image" class="img-responsive" src="https://static.igem.org/mediawiki/2016/d/d2/T--SCAU-China--Engagement5.png">
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<div class="p_font_size">We hoped our human practice work can influence more ordinary people.So,above all,we should realize other people's opinion to transgenic food.Because of that,we made a questionnaire about GMO.</div>
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<div class="p_font_size">The data we’ve obtained in questionnaire is showing below:</div>
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<div class="p_font_size">The respondents’  proportion of men and women is approximately balanced. The number of age group which between 18 from 25 is the majority. What’s more , the respondents  have a Bachelor degree or a higher degree.</div>
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<div class="h3_font_size">1.1 The main content</div>
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<div class="p_font_size">Question 4: Do you know what is Genetically modified (gm)?</div>
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<img alt="image" class="img-responsive" src="https://static.igem.org/mediawiki/2016/8/85/T--SCAU-China--Engagement7.jpg">
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<div class="p_font_size">Analysis of the Q4:4/5 of the respondents have a certain understanding for "GM technology"，1/5 respondents don’t understand this technology or even don’t know. This suggests that the "GM technology" was received by the majority of respondents.</div>
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<div class="p_font_size">Question 6:Will you eat gm food?</div>
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<img alt="image" class="img-responsive" src="https://static.igem.org/mediawiki/2016/0/08/T--SCAU-China--Engagement8.jpg">
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<img alt="image" class="img-responsive" src="https://static.igem.org/mediawiki/2016/9/99/T--SCAU-China--Engagement9.png">
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</div>
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<div class="p_font_size">Analysis :About a third of the people can accept genetically modified (gm) food. 17% of people determined not to eat genetically modified food.Suggest that the public acceptance for genetically modified foods is improving.</div>
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<div class="p_font_size">Question 11:  Is transgenosis beneficial or harmful in your opinions?</div>
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<div class="row" style="margin-top:20px">
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<div class="col s12" align="center">
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<img alt="image" class="img-responsive" src="https://static.igem.org/mediawiki/2016/9/96/T--SCAU-China--Engagement10.jpg">
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<div class="col s10" align="center">
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<img alt="image" class="img-responsive" src="https://static.igem.org/mediawiki/2016/6/62/T--SCAU-China--Engagement11.png">
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<div class="row" style="margin-top:20px">
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<img alt="image" class="img-responsive" src="https://static.igem.org/mediawiki/2016/4/4c/T--SCAU-China--Engagement12.png">
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<div class="p_font_size">Analysis : Most people had skeptical attitude to genetically modification (gm) . For respondents,genetically modification is an emerging technology. The public don’t understand enough about genetically modified crops,so they cannot hold a certain attitude to it. </div>
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<div class="p_font_size">Question 12 :Will you use gm products (non-food)?</div>
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<div class="col s1"></div>
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<img alt="image" class="img-responsive" src="https://static.igem.org/mediawiki/2016/2/29/T--SCAU-China--Engagement13.png">
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<div class="p_font_size">Question13: Have you ever heard of skin care product that contains astaxanthin?</div>
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<div class="row" style="margin-top:20px">
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<div class="col s12" align="center">
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<img alt="image" class="img-responsive" src="https://static.igem.org/mediawiki/2016/a/ab/T--SCAU-China--Engagement14.jpg">
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<img alt="image" class="img-responsive" src="https://static.igem.org/mediawiki/2016/a/a1/T--SCAU-China--Engagement15.png">
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</div>
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<div class="p_font_size">Question 14:Will you choose skin care product or health food contains astaxanthin (gm) ？</div>
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      <div class="text">The parts we submit are listed at the table below.<br><br>
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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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    <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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    <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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  <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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<script src="https://2016.igem.org/Team:SCAU-China/jsfile?action=raw&ctype=text/javascript"></script>  <!--mater-->
  
  
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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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  (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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  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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  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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  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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  Zeaxanthin + 2 Oxygen → Astaxanthin + 2H2O
 
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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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      <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 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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      <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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      <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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      <p><img src="https://static.igem.org/mediawiki/2016/4/4b/T--SCAU-China--part6.png"></p>
 
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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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      <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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    <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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    <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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      <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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      <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 13:23, 14 October 2016

SCAU

Gold
Our human practice work involved several questions relating to bio-safety,social justice and public acceptance.Our astarice is actually a genetically modified organism,so our work focused on people’s concern about GMO.
1. Investigation
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We hoped our human practice work can influence more ordinary people.So,above all,we should realize other people's opinion to transgenic food.Because of that,we made a questionnaire about GMO.
The data we’ve obtained in questionnaire is showing below:
The respondents’ proportion of men and women is approximately balanced. The number of age group which between 18 from 25 is the majority. What’s more , the respondents have a Bachelor degree or a higher degree.
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1.1 The main content
Question 4: Do you know what is Genetically modified (gm)?
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Analysis of the Q4:4/5 of the respondents have a certain understanding for "GM technology"，1/5 respondents don’t understand this technology or even don’t know. This suggests that the "GM technology" was received by the majority of respondents.
Question 6:Will you eat gm food?
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Analysis :About a third of the people can accept genetically modified (gm) food. 17% of people determined not to eat genetically modified food.Suggest that the public acceptance for genetically modified foods is improving.
Question 11: Is transgenosis beneficial or harmful in your opinions?
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Analysis : Most people had skeptical attitude to genetically modification (gm) . For respondents,genetically modification is an emerging technology. The public don’t understand enough about genetically modified crops,so they cannot hold a certain attitude to it.
Question 12 :Will you use gm products (non-food)?
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Question13: Have you ever heard of skin care product that contains astaxanthin?
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Question 14:Will you choose skin care product or health food contains astaxanthin (gm) ？