Team:Harvard/vectors/vectors

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<h1>meet the vectors</h1>
<h1>meet the vectors</h1>
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<p>Our vectors are a modification of the pORE agrobacterium vector system developed by Coutu et al at Agriculture and Agrofood Canada. This vector system is designed for agrobacterium mediated plant transformation, and is a binary system with the vector containing the site for foreign constructs and the appropriate flanking regions to be recognized for introduction to the plant. The virulence genes are present on a separate vector contained in the agrobacterium, separate from the TDNA.</p>
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<p>Our vectors are a modification of the pORE agrobacterium vector system developed by Coutu, ''et al'', at Agriculture and Agrofood Canada, which we obtained from <a href="http://www.arabidopsis.org/">The Arabidopsis Information Resource (TAIR)</a>. This vector system is designed for agrobacterium mediated plant transformation, and is a binary system with the vector containing the site for foreign constructs and the appropriate flanking regions to be recognized for introduction to the plant. The virulence genes are present on a separate vector contained in the agrobacteria, separate from the TDNA.</p>
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<p>To modify the pORE series binary vectors to fit the biobrick standard, we digested the vectors with. We worked with two of each of the open, reporter, and expression series vectors, creating a total of six biobrick standard vectors suitable for agrobacterium mediated plant transformation.</p>
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<p>To modify the pORE series binary vectors to fit the BioBrick standard, we used a combination of PCR mutagenesis and digestion strategies to replace the vectors' existing multiple cloning site with the BioBrick multiple cloning site. We worked with two of each of the open, reporter, and expression series vectors, creating a total of six BioBrick vectors suitable for agrobacterium mediated plant transformation.</p>
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        <li><a href="#open"><span>Open Series</span></a></li>
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        <li><a href="#reporter"><span>Reporter Series</span></a></li>
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        <li><a href="#expression"><span>Expression Series</span></a></li>
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<div>open series &nbsp; <a href="https://static.igem.org/mediawiki/2010/9/9f/Open_Large.png" id="single_image" style="font-size:12px">click to enlarge</a></div><hr/>
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<p style="padding:10px"><br/>The open series vectors are designed for general insertion of a construct. We modified the vectors pORE O1 and pORE O2. Each has a different antibiotic resistance.</p></td></tr></table>
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      <p>In our second scheme, we will knock out BETA-OHASE 1 on its own. We hope that this will induce accumulation of beta-carotene.</p><br/><br/>
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      <p>In our third scheme, we will knock out both LUT2 and BETA-OHASE1. We hope that this will induce accumulation of some lycopene and some beta-carotene.</p><br/>
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<p style="padding:10px"><br/>The open series vectors are designed for general insertion of a construct. We modified the vectors pORE O1 and pORE O2. Each has a different antibiotic resistance.</p></td>
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<p style="padding:10px"><br/>The open series vectors are designed for general insertion of a construct. We modified the vectors pORE O1 and pORE O2. pORE O1 confers plant resistance to <a href="http://en.wikipedia.org/wiki/Glufosinate">glufosinate</a>, and pORE O2 confers plant resistance to <a href="http://en.wikipedia.org/wiki/Kanamycin">kanamycin</a>, to be used in transformant selection.</p></td>
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<td style="vertical-align:top"><p style="padding:10px"><br/>The reporter series vectors contain a reporter on the trailing end of the multiple cloning site such that expression of the reporter follows that of the inserted construct. We modified the vectors pORE R1 and pORE R3. pORE R1 contains the gusA reporter, and pORE R3 the smgfp reporter. </p></td>
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<td style="vertical-align:top"><p style="padding:10px"><br/>The reporter series vectors contain a reporter on the trailing end of the multiple cloning site such that expression of the reporter follows that of the inserted construct. We modified the vectors pORE R1 and pORE R3. pORE R1 contains the gusA reporter, and pORE R3 the smgfp reporter. Both vectors confer plant resistance to kanamycin. </p></td>
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<td style="vertical-align:top"><p style="padding:10px"><br/>The expression series vectors contain a promoter preceding the multiple cloning site such that the inserted construct can be easily expressed through activation of the contained promoter. We modified the vectors pORE E3 and pORE E4. Both contain the ENTCUP2 promoter, though each has a different plant resistance gene (E3 contains pat and E4 nptIII)</p></td>
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<td style="vertical-align:top"><p style="padding:10px"><br/>The expression series vectors contain a promoter preceding the multiple cloning site such that the inserted construct can be easily expressed through activation of the contained promoter. We modified the vectors pORE E3 and pORE E4. Both vectors contain the ENTCUP2 promoter. pORE E3 confers plant resistance to glufosinate, and pORE E4 confers plant resistance to kanamycin</p></td>
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Source: Coutu, Catherine et al. "pORE: a modular binary vector series suited for both
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monocot and dicot plant transformation." <i>Transgenic Res</i> (2007) 16:771–781.
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Latest revision as of 21:37, 24 October 2010



meet the vectors

Our vectors are a modification of the pORE agrobacterium vector system developed by Coutu, ''et al'', at Agriculture and Agrofood Canada, which we obtained from The Arabidopsis Information Resource (TAIR). This vector system is designed for agrobacterium mediated plant transformation, and is a binary system with the vector containing the site for foreign constructs and the appropriate flanking regions to be recognized for introduction to the plant. The virulence genes are present on a separate vector contained in the agrobacteria, separate from the TDNA.

To modify the pORE series binary vectors to fit the BioBrick standard, we used a combination of PCR mutagenesis and digestion strategies to replace the vectors' existing multiple cloning site with the BioBrick multiple cloning site. We worked with two of each of the open, reporter, and expression series vectors, creating a total of six BioBrick vectors suitable for agrobacterium mediated plant transformation.

open series   click to enlarge


The open series vectors are designed for general insertion of a construct. We modified the vectors pORE O1 and pORE O2. pORE O1 confers plant resistance to glufosinate, and pORE O2 confers plant resistance to kanamycin, to be used in transformant selection.
reporter series   click to enlarge


The reporter series vectors contain a reporter on the trailing end of the multiple cloning site such that expression of the reporter follows that of the inserted construct. We modified the vectors pORE R1 and pORE R3. pORE R1 contains the gusA reporter, and pORE R3 the smgfp reporter. Both vectors confer plant resistance to kanamycin.

expression series   click to enlarge


The expression series vectors contain a promoter preceding the multiple cloning site such that the inserted construct can be easily expressed through activation of the contained promoter. We modified the vectors pORE E3 and pORE E4. Both vectors contain the ENTCUP2 promoter. pORE E3 confers plant resistance to glufosinate, and pORE E4 confers plant resistance to kanamycin
Source: Coutu, Catherine et al. "pORE: a modular binary vector series suited for both monocot and dicot plant transformation." Transgenic Res (2007) 16:771–781.





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