Difference between revisions of "Team:HebrewU/Plant"

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             <li style="float:left;margin-top:12px;padding-left:15px;"><br />Hebrew</li>
 
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             <li><a href="https://2018.igem.org/Team:HebrewU/Results">Results</a></li>
 
             <li><a href="https://2018.igem.org/Team:HebrewU/Results">Results</a></li>
 
             <li><a href="https://2018.igem.org/Team:HebrewU/Parts">Parts</a></li>
 
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             <li><a href="https://2018.igem.org/Team:HebrewU/Motivation">Moolti</a></li>
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  <h1 class="w3-margin w3-jumbo">Plant Design</h1>
 
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        <p style="padding-left:150px;padding-right:150px;text-align:justify;line-height:1.5">
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        Our teams pathway was designed for plants. Despite analyzing the pathway in yeast, we always saw plants as our final frontier. On the <a href="https://2018.igem.org/Team:HebrewU/Open_Source">open source page</a>, we discuss plants such as pumpkins and wheat that are ideal for real world application of our project, as well as mechanisms for sterilizing GMO plants. Sterilizing plants ensures that cross pollination between GMOs and natural fauna cannot happen- allowing for the responsible implementation of our project. Working with the plants native features, utilizing roots for uptake and native enzymes we created a synergy between natural and synthetic biology. The engineered pathway ends with a product (pyrocatechol) which seamlessly enters plants metabolism via Polyphenol Oxidase enzymes, found in almost all plants. <sup>1</sup>
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      The initial technologies for engineering the agrobacterium involved complex microbial genetic methodologies that inserted the gene of interest into the transfer DNA (T-DNA) region of the Ti-plasmids. However, as these technologies developed, and more research was done, biologists realized that the T-DNA genes did not have to sit on the same plasmid as the Vir (virulence) genes to successfully introduce genes in to plants. Thus, the Binary Vector method was created, and this once big and complicated vector was split in two, easy to work with vectors. This binary system permitted simple manipulation of Agrobacterium and opened up the field of plant genetic engineering. It is composed of the borders of T-DNA, multiple cloning sites, replication functions for E. coli and A. tumefaciens, selectable marker genes, reporter genes, and other accessory elements that can improve the efficiency of and give further capability to the system.<sup>2,3,4</sup>
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        We used the pHGHPB vector which contains all of the above mentioned features. In this vector, GFP and the inserted gene are fused by a Poly-A Bridge, creating a hybrid protein. Using this method, its ensured, that were ever we see GFP expression, we know that our protein is be expressed as well. In addition, Basta antibiotic resistance gene is expressed, allowing for easier identification of transgenic plants when grown on appropriate media. Both of these genes are regulated by the 35S promoter, derived from cauliflower mosaic virus, which is a constitutive promoter, active in all tissues of the plant.
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We cloned multiple vectors with our genes, but only transformed plants with Haloacid Dehalogenase. As each generation of Arabidopsis takes approximately 3 months to grow + transform, it was not possible to transform a plant with our entire pathway in the time we had for this year's competition. As such we decided to use the Dehalogenase, even though it does not completely breakdown the molecule, it partially dechlorinates TCDD, reducing its toxicity greatly. <br />  <br />   
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            <img src="https://static.igem.org/mediawiki/2018/3/38/T--hebrewu--Plant_Des2.png" width="35%">
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        <h2 style="padding-left:150px;padding-right:150px;text-align:justify;line-height:1.5"> References: </h2>
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                1. <a hred="https://www.agilent.com/cs/library/usermanuals/public/217451.pdf"> [Reference 1] </a><br />
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                2. <a hred="https://www.neb.com/-/media/catalog/datacards-or-manuals/manuale2611.pdf">[Reference 1]</a><br />
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            3. <a hred="https://www.neb.com/-/media/catalog/datacards-or-manuals/manuale2611.pdf">[Reference 1]</a><br />
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              4. <a hred="https://www.neb.com/-/media/catalog/datacards-or-manuals/manuale2611.pdf">[Reference 1]</a><br />
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Revision as of 20:23, 13 October 2018

HebrewU HujiGEM 2018





Our teams pathway was designed for plants. Despite analyzing the pathway in yeast, we always saw plants as our final frontier. On the open source page, we discuss plants such as pumpkins and wheat that are ideal for real world application of our project, as well as mechanisms for sterilizing GMO plants. Sterilizing plants ensures that cross pollination between GMOs and natural fauna cannot happen- allowing for the responsible implementation of our project. Working with the plants native features, utilizing roots for uptake and native enzymes we created a synergy between natural and synthetic biology. The engineered pathway ends with a product (pyrocatechol) which seamlessly enters plants metabolism via Polyphenol Oxidase enzymes, found in almost all plants. 1



The initial technologies for engineering the agrobacterium involved complex microbial genetic methodologies that inserted the gene of interest into the transfer DNA (T-DNA) region of the Ti-plasmids. However, as these technologies developed, and more research was done, biologists realized that the T-DNA genes did not have to sit on the same plasmid as the Vir (virulence) genes to successfully introduce genes in to plants. Thus, the Binary Vector method was created, and this once big and complicated vector was split in two, easy to work with vectors. This binary system permitted simple manipulation of Agrobacterium and opened up the field of plant genetic engineering. It is composed of the borders of T-DNA, multiple cloning sites, replication functions for E. coli and A. tumefaciens, selectable marker genes, reporter genes, and other accessory elements that can improve the efficiency of and give further capability to the system.2,3,4



We used the pHGHPB vector which contains all of the above mentioned features. In this vector, GFP and the inserted gene are fused by a Poly-A Bridge, creating a hybrid protein. Using this method, its ensured, that were ever we see GFP expression, we know that our protein is be expressed as well. In addition, Basta antibiotic resistance gene is expressed, allowing for easier identification of transgenic plants when grown on appropriate media. Both of these genes are regulated by the 35S promoter, derived from cauliflower mosaic virus, which is a constitutive promoter, active in all tissues of the plant.

We cloned multiple vectors with our genes, but only transformed plants with Haloacid Dehalogenase. As each generation of Arabidopsis takes approximately 3 months to grow + transform, it was not possible to transform a plant with our entire pathway in the time we had for this year's competition. As such we decided to use the Dehalogenase, even though it does not completely breakdown the molecule, it partially dechlorinates TCDD, reducing its toxicity greatly.



References:

1. [Reference 1]
2. [Reference 1]
3. [Reference 1]
4. [Reference 1]