Difference between revisions of "Team:SCAU-China/Basic Part"

 
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    <li><a href="https://2016.igem.org/Team:SCAU-China/Time_shaft">Time shaft</a></li>
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<div class="h1_font_size">Parts</div>
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<div class="h2_font_size">1. Parts Table</div>
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<div class="p_font_size" style="text-indent:0em">The parts we submitted are listed at the table below.</div>
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<th>Part name</th>
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<th>Description</th>
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<th>Length</th>
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<td><a href="http://parts.igem.org/Part:BBa_K1959000" text-decoration=underline>BBa_K1959000</a></td>
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<td><em>PSY </em>CDS codon optimized for <em>O. sativa.</em></td>
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<td>1193 bp</td>
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</tr>
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<tr>
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<td><a href="http://parts.igem.org/Part:BBa_K1959001" text-decoration=underline>BBa_K1959001</a></td>
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<td><em>CrtI</em> CDS codon optimized for <em>O. sativa.</em></td>
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<td>1650 bp</td>
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</tr>
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  <tr>
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<td><a href="http://parts.igem.org/Part:BBa_K1959002" text-decoration=underline>BBa_K1959002</a></td>
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<td><em>BHY</em> CDS codon optimized for <em>O. sativa.</em></td>
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<td>1053 bp</td>
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</tr>
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<tr>
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<td><a href="http://parts.igem.org/Part:BBa_K1959003" text-decoration=underline>BBa_K1959003</a></td>
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<td><em>BKT </em>CDS codon optimized for <em>O. sativa.</em></td>
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<td>1158 bp</td>
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</tr>
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</tbody>
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</table>
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<br>
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<div class="h2_font_size">2. Origin and functions of the part</div>
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<div class="p_font_size">(1) BBa_K195900</div>
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<br>
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<div class="p_font_size">This part is the coding sequence (CDS) of phytoene synthase (<font style="font-style:italic">PSY</font>) from <em>Zea mays</em>, which is codon optimized for expression in rice<em> (Oryza sativa)</em>. <font style="font-style:italic">PSY</font> is part of β-carotene biosynthesis pathway and it catalyzes the conversion of geranylgeranyl diphosphate to phytoene.</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/d/d2/T--SCAU-China--Part8.png" width="1000px">
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<br>
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<div class="p_font_size">(2) BBa_K1959001</div>
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<br>
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<div class="p_font_size">This part is the phytoene desaturase (<font style="font-style:italic">CrtI</font>) from<em> Erwinia uredovora</em> fused with<em> Pea </em>transit peptide. It is codon optimized for rice. Bacterial phytoene desaturase catalyzes conversion of phytoene to lycopene.</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/2016/2/2d/T--SCAU-China--Part2.png" width="1000px">
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<br>
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<div class="p_font_size">(3) BBa_K1959002 </div>
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<br>
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<div class="p_font_size">This part is the β-carotene hydroxylase (<font style="font-style:italic">BHY</font>) coding sequence from <em>Haematococcus pluvialis</em>. The <em>Pea</em> transit peptide is functionally fused with <font style="font-style:italic">BHY</font> in order to help <font style="font-style:italic">BHY</font> polypeptide folding correctly. <font style="font-style:italic">BHY</font> sequence is codon optimized for expression in rice. The <font style="font-style:italic">BHY</font> is part of astaxanthin biosynthesis pathway and it catalyzes conversion of β-carotene to zeaxanthin (an astaxanthin precursor).</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/d/dc/T--SCAU-China--Part3.png" width="1000px">
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<br>
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<div class="p_font_size">(4) BBa_K1959003 </div>
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<br>
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<div class="p_font_size">This part contains <em>Pea </em>transit peptide and coding sequence of β-carotene ketolase (<font style="font-style:italic">BKT</font>). <font style="font-style:italic">BKT</font> catalyzes zeaxanthin to astaxanthin. This sequence is from <em>Chlamydomonas reinhardti,</em> and codon optimization has been made for stable expression in rice.</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/2016/3/32/T--SCAU-China--Part4.png" width="1000px">
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</div>
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</div>
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<div class="p_font_size">When the above four foreign genes were stacked into rice genome, under the control of endosperm-specific promoters, the functional astaxanthin biosynthetic pathway(see figure below) was reconstructed in rice endosperm.</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/2016/6/66/T--SCAU-China--Design9.png" width="800px">
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</div>
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</div>
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<div class="p_font_size"style="margin-bottom:20px"><small>The reconstruction of astaxanthin biosynthetic pathway in the endosperm. β-LYC (β-lycopene cyclase) is a endogenous gene of rice.</small></div>
 +
<br>
 +
<div class="p_font_size">The genes described above were assembled in a transformation-competent artificial chromosome (TAC)-based binary plasmid and introduced into rice genome using <em>Agrobacterium</em>-mediated method. </div>
 +
<br>
 +
<div class="p_font_size">The presences of these genes were confirmed by PCR analyses and the expression of the transgenes in rice endosperm was verified by RT-PCR and qRT-PCR.</div>
 +
<br>
 +
<div class="p_font_size">Orange-red rice seeds could be easily observed by naked eyes and the products of astaxanthin was detected in the extraction of rice seeds by HPLC analyses. Furthermore, the HPLC profile shows that astaxanthin was predominant composition of the total carotenoids in rice endosperm. All of the experimental results proved that we successfully reconstructed the functional astaxanthin biosynthesis pathway in rice endosperm. </div>
 +
<br>
 +
<div class="p_font_size">Our transgenic results fully proved that above parts are functional components for astaxanthin biosynthesis. These functional parts can be further used in synthetic biology of other plants.</div>
 +
<br>
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<div class="p_font_size">For more details, please refer to our <a href="https://2016.igem.org/Team:SCAU-China/Experiments" >RESULTS</a> section.</div>
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A <b>basic part</b> is a functional unit of DNA that cannot be subdivided into smaller component parts. <a href="http://parts.igem.org/wiki/index.php/Part:BBa_R0051">BBa_R0051</a> is an example of a basic part, a promoter regulated by lambda cl.
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<p>Most genetically-encoded functions have not yet been converted to BioBrick parts. Thus, there are <b>many</b> opportunities to find new, cool, and important genetically encoded functions, and refine and convert the DNA encoding these functions into BioBrick standard biological parts. </p>
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<h4>Note</h4>
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<p>This page should list all the basic parts your team has made during your project. You must add all characterization information for your parts on the Registry. You should not put characterization information on this page.</p>
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Latest revision as of 09:42, 17 October 2016

SCAU

Parts
1. Parts Table
The parts we submitted are listed at the table below.
Part name Description Length
BBa_K1959000 PSY CDS codon optimized for O. sativa. 1193 bp
BBa_K1959001 CrtI CDS codon optimized for O. sativa. 1650 bp
BBa_K1959002 BHY CDS codon optimized for O. sativa. 1053 bp
BBa_K1959003 BKT CDS codon optimized for O. sativa. 1158 bp

2. Origin and functions of the part
(1) BBa_K195900

This part is the coding sequence (CDS) of phytoene synthase (PSY) from Zea mays, which is codon optimized for expression in rice (Oryza sativa). PSY is part of β-carotene biosynthesis pathway and it catalyzes the conversion of geranylgeranyl diphosphate to phytoene.
image

(2) BBa_K1959001

This part is the phytoene desaturase (CrtI) from Erwinia uredovora fused with Pea transit peptide. It is codon optimized for rice. Bacterial phytoene desaturase catalyzes conversion of phytoene to lycopene.
image

(3) BBa_K1959002

This part is the β-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 rice. The BHY is part of astaxanthin biosynthesis pathway and it catalyzes conversion of β-carotene to zeaxanthin (an astaxanthin precursor).
image

(4) BBa_K1959003

This part contains Pea transit peptide and coding sequence of β-carotene ketolase (BKT). BKT catalyzes zeaxanthin to astaxanthin. This sequence is from Chlamydomonas reinhardti, and codon optimization has been made for stable expression in rice.
image
When the above four foreign genes were stacked into rice genome, under the control of endosperm-specific promoters, the functional astaxanthin biosynthetic pathway(see figure below) was reconstructed in rice endosperm.
image
The reconstruction of astaxanthin biosynthetic pathway in the endosperm. β-LYC (β-lycopene cyclase) is a endogenous gene of rice.

The genes described above were assembled in a transformation-competent artificial chromosome (TAC)-based binary plasmid and introduced into rice genome using Agrobacterium-mediated method.

The presences of these genes were confirmed by PCR analyses and the expression of the transgenes in rice endosperm was verified by RT-PCR and qRT-PCR.

Orange-red rice seeds could be easily observed by naked eyes and the products of astaxanthin was detected in the extraction of rice seeds by HPLC analyses. Furthermore, the HPLC profile shows that astaxanthin was predominant composition of the total carotenoids in rice endosperm. All of the experimental results proved that we successfully reconstructed the functional astaxanthin biosynthesis pathway in rice endosperm.

Our transgenic results fully proved that above parts are functional components for astaxanthin biosynthesis. These functional parts can be further used in synthetic biology of other plants.

For more details, please refer to our RESULTS section.