Difference between revisions of "Team:SCAU-China/Proof"

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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/Proof">Proof</a></li>
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<li><a href="https://2016.igem.org/Team:SCAU-China/Demonstrate">Demonstrate</a></li>
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<div class="h1_font_size">Proof</div>
  
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<div class="h2_font_size">Analysis of transgenes</div>
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<div class="p_font_size"  style="text-indent:0em" >After <font style="font-style:italic">Agrobacterium</font>-mediated rice calli transformation, several hygromycin resistant rice lines were obtained, and their further molecular analysis were done. The genomic DNA of T<SUB>0</SUB> transformants were extracted to do PCR amplifying HPT and Cre. Then PCR positive lines were selected to detect four foreign genes <font style="font-style:italic">(PSY, CrtI, BHY </font>and<font style="font-style:italic"> BKT)</font>. The PCR results of some lines showed the expected bands of four genes were detected (Figure 3).</div>
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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2016.igem.org/Judging/Medals">gold medal criterion for proof of concept</a>. </p>
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<p> Delete this box in order to be evaluated for this medal. See more information at <a href="https://2016.igem.org/Judging/Pages_for_Awards/Instructions"> Instructions for Pages for awards</a>.</p>
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<div class="p_font_size" style="margin-bottom:20px"><small> <font style="font-weight:bold">Figure 3</font> &nbsp;&nbsp;PCR assays of several transgenic rice lines. M, Marker 2K plus. CK+, positive control (plasmid pYLTAC380MF-BBPC). WT, negative control (wild-type rice cultivar HG1).</small></div>
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<div class="p_font_size">To detect expression levels of these four foreign genes involved in astaxanthin biosynthesis, total RNA of transgenic rice seeds were extracted and their cDNA was synthesized from 1 μg of DNase-treated RNA. The results of RT-PCR showed the expected bands of the four key genes for synthesizing astaxanthin in transgenic rice seeds (Figure 4).</div>
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<div class="p_font_size" style="margin-bottom:20px"><small> <font style="font-weight:bold">Figure 4</font> &nbsp;&nbsp;RT-PCR analysis of expression levels of <font style="font-style:italic">PSY, CrtI, BKY, </font>and <font style="font-style:italic">BHY</font> genes in several transgenic rice. Rice <font style="font-style:italic">OsActin1</font> was as an internal control. CK+, positive control (plasmid pYLTAC380MF-BBPC). WT, negative control (wild-type rice cultivar HG1).</small></div>
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<div class="p_font_size">These results of PCR and RT-PCR demonstrated that we successfully obtained transgenic rice with all of four stacking genes <font style="font-style:italic">(PSY, CrtI, BHY </font>and<font style="font-style:italic"> BKT). </font></div>
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<p>&nbsp;</p>
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<div class="p_font_size">In summary, we successfully obtained transformants with all of transgenes in lab and detected obvious transcriptional activity in endosperm. These results indicated that our designed pathway was realizable in rice endosperm. To further confirm astaxanthin production, several analysis were carried out. The results were shown in <a href="https://2016.igem.org/Team:SCAU-China/Demonstrate" text-decoration=underline>Demonstrate page</a>.</div>
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iGEM teams are great at making things work! We value teams not only doing an incredible job with theoretical models and experiments, but also in taking the first steps to make their project real.  
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<h4> What should we do for our proof of concept? </h4>
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You can assemble a device from BioBricks and show it works. You could build some equipment if you're competing for the hardware award. You can create a working model of your software for the software award. Please note that this not an exhaustive list of activities you can do to fulfill the gold medal criterion. As always, your aim is to impress the judges!
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Latest revision as of 05:33, 18 October 2016

SCAU

Proof
Analysis of transgenes
After Agrobacterium-mediated rice calli transformation, several hygromycin resistant rice lines were obtained, and their further molecular analysis were done. The genomic DNA of T0 transformants were extracted to do PCR amplifying HPT and Cre. Then PCR positive lines were selected to detect four foreign genes (PSY, CrtI, BHY and BKT). The PCR results of some lines showed the expected bands of four genes were detected (Figure 3).
image
Figure 3   PCR assays of several transgenic rice lines. M, Marker 2K plus. CK+, positive control (plasmid pYLTAC380MF-BBPC). WT, negative control (wild-type rice cultivar HG1).
To detect expression levels of these four foreign genes involved in astaxanthin biosynthesis, total RNA of transgenic rice seeds were extracted and their cDNA was synthesized from 1 μg of DNase-treated RNA. The results of RT-PCR showed the expected bands of the four key genes for synthesizing astaxanthin in transgenic rice seeds (Figure 4).
image
Figure 4   RT-PCR analysis of expression levels of PSY, CrtI, BKY, and BHY genes in several transgenic rice. Rice OsActin1 was as an internal control. CK+, positive control (plasmid pYLTAC380MF-BBPC). WT, negative control (wild-type rice cultivar HG1).
These results of PCR and RT-PCR demonstrated that we successfully obtained transgenic rice with all of four stacking genes (PSY, CrtI, BHY and BKT).

 

In summary, we successfully obtained transformants with all of transgenes in lab and detected obvious transcriptional activity in endosperm. These results indicated that our designed pathway was realizable in rice endosperm. To further confirm astaxanthin production, several analysis were carried out. The results were shown in Demonstrate page.