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

 
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<div class="h1_font_size">Experiement</div>
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<div class="h1_font_size">Experiments</div>
  
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<div class="h2_font_size">Vector construction</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">To synthesize astaxanthin and delete selective resistance 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.</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>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 Isites. (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="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 Agrobacterium tumefaciens strain EHA105 by electroporation. Then the Agrobacterium cells were cocultured 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. You can read more details, <a href="https://2016.igem.org/Team:SCAU-China/Protocol#rice_transformation" text-decoration=underline>please click here!</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">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>Figure 2 Agrobacterium-mediated rice calli transformation process.</small></div>
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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>
 
<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).-->
 
<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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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.