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

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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/Members">Members</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/Protocol">Protocol</a></li>
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<div class="h1_font_size">Experiments</div>
  
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<div class="h2_font_size"><font style="font-weight:bold">Vector construction</font></div>
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<div class="p_font_size"  style="text-indent:0em" >To synthesize astaxanthin and delete antibiotic resistance selection marker gene, six gene expression cassettes were assembled into a TAC-based binary acceptor vector, designated as pYLTAC380MF-BBPC (Figure 1), by multiple rounds of gene assembly cycles using our marker-free TransGene Stacking II system, in which contained four genes (<font style="font-style:italic">CrtI, PSY, BKT </font>and <font style="font-style:italic">BHY</font>) under the control of 4 different endosperm-specific promoters for astaxanthin biosynthesis, and two genes (<font style="font-style:italic">HPT</font> and <font style="font-style:italic">Cre</font>) for marker-free deletion.</div>
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<div class="p_font_size" style="margin-bottom:20px"><small><font style="font-weight:bold">Figure 1</font> &nbsp;&nbsp;Structure and restriction analysis of multigene recombinant constructs. Structure and restriction analysis of recombinant constructs. (A) Structure of a construct pYLTAC380MF-BBPC containing four genes for astaxanthin biosynthesis and two genes for marker free. The numbers in parentheses indicate the order of the sequences inserting into the vector. N denotes Not I sites. (B) Restriction analysis of a series of multigene constructs containing different numbers of genes (from lanes 3 to 6). Arrows indicate the different insertion genes.</small></div>
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<div class="h2_font_size"><font style="font-weight:bold">Rice transformation</font></div>
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<div class="p_font_size">The multigene construct pYLTAC380MF-BBPC was introduced into <font style="font-style:italic">Agrobacterium tumefaciens</font> strain <font style="font-style:italic">EHA105</font> by electroporation. Then the <font style="font-style:italic">Agrobacterium</font> cells were co-cultured with embryogenic calli induced from mature seeds of <font style="font-style:italic">Indica</font> rice varieties Huaguang1 (HG1) as described (Lin et al., PNAS, 2003, 100: 5962-5967). Regenerating calli were selected in the presence of 50 mg/L hygromycin and subsequently transferred to rooting medium containing hygromycin. After further culturing for 3 to 4 weeks, transformed plants were transferred to soil in a greenhouse. Figure 2 showed the <font style="font-style:italic">Agrobacterium</font>-mediated transformation process and transgenic rice cultivation. If you want to read more details, you can click here to see our <a href="https://2016.igem.org/Team:SCAU-China/Protocol#rice_transformation" text-decoration=underline>protocol</a> and <a href="https://2016.igem.org/Team:SCAU-China/Notebook" text-decoration=underline>notebook!</a></div><!--(加链接到Notebook栏目下面的3.protocol的2. rice transformation)-->
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<div class="p_font_size" style="margin-bottom:20px"><small> <font style="font-weight:bold">Figure 2</font> &nbsp;&nbsp;The proccess of <font style="font-style:italic">Agrobacterium</font>-mediated transformation in rice.</small></div>
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<div class="p_font_size">To confirm the realization of our designed pathway, PCR amplification and semi-quantitative RT-RCR analysis were performed. The experimental results were presented in our <a href="https://2016.igem.org/Team:SCAU-China/Proof" text-decoration=underline>Proof page.</a></div><!--(超链接到 Proof).-->
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<p>Describe the experiments, research and protocols you used in your iGEM project.</p>
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<h5>What should this page contain?</h5>
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<li> Protocols </li>
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<li> Experiments </li>
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<li>Documentation of the development of your project </li>
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<h5>Inspiration</h5>
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<li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
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<li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
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<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
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Latest revision as of 05:31, 18 October 2016

SCAU

Experiments
Vector construction
To synthesize astaxanthin and delete antibiotic resistance selection marker gene, six gene expression cassettes were assembled into a TAC-based binary acceptor vector, designated as pYLTAC380MF-BBPC (Figure 1), by multiple rounds of gene assembly cycles using our marker-free TransGene Stacking II system, in which contained four genes (CrtI, PSY, BKT and BHY) under the control of 4 different endosperm-specific promoters for astaxanthin biosynthesis, and two genes (HPT and Cre) for marker-free deletion.
image
Figure 1   Structure and restriction analysis of multigene recombinant constructs. Structure and restriction analysis of recombinant constructs. (A) Structure of a construct pYLTAC380MF-BBPC containing four genes for astaxanthin biosynthesis and two genes for marker free. The numbers in parentheses indicate the order of the sequences inserting into the vector. N denotes Not I sites. (B) Restriction analysis of a series of multigene constructs containing different numbers of genes (from lanes 3 to 6). Arrows indicate the different insertion genes.
Rice transformation
The multigene construct pYLTAC380MF-BBPC was introduced into Agrobacterium tumefaciens strain EHA105 by electroporation. Then the Agrobacterium cells were co-cultured with embryogenic calli induced from mature seeds of Indica rice varieties Huaguang1 (HG1) as described (Lin et al., PNAS, 2003, 100: 5962-5967). Regenerating calli were selected in the presence of 50 mg/L hygromycin and subsequently transferred to rooting medium containing hygromycin. After further culturing for 3 to 4 weeks, transformed plants were transferred to soil in a greenhouse. Figure 2 showed the Agrobacterium-mediated transformation process and transgenic rice cultivation. If you want to read more details, you can click here to see our protocol and notebook!
image
Figure 2   The proccess of Agrobacterium-mediated transformation in rice.
To confirm the realization of our designed pathway, PCR amplification and semi-quantitative RT-RCR analysis were performed. The experimental results were presented in our Proof page.