Team:WashU/DesignSynecho

From 2012.igem.org

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__NOTOC__
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=Our Design=
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<br><br><br>
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==The Gene==
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https://lh3.googleusercontent.com/-V1CC044VgSY/T9oz9McUKFI/AAAAAAAAAFk/DzPhWQ7CX-M/s800/gene.jpg
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==Exposition==
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<div id = "design">
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<h1>Phase II</h1>
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<h3>Design</h3>
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The purpose of phase II is to optimize the expression of the proteins necessary for the production of crocin glycosides and safranal.  Unfortunately, ZCD has been shown to produce inclusion bodies when expressed'' in vivo'' in ''E. coli'' and so an alternative method needed to be developed to create a functional protein.  This was done by following the technique described by Florence Bouvier (The Plant Cell, vol. 15 47-62).
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<img src="https://static.igem.org/mediawiki/igem.org/2/2f/Thiofusion_diagram_2.jpg" ALIGN=RIGHT width="786" border="0" usemap="#map" >
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The first thing we did was figuring out the carotenoid biochemical pathway from beta carotene to our desired products safranal, crocin and picrocrocin. We noticed that the pathway was very complex and lengthy. Therefore we wanted to simplify things by finding an organism that endogenously produced one of the intermediates close to our final products. Our advisers suggested using Synechocystis PCC 6803, which produced beta carotene and many of its derivatives including Zeaxanthin which Crocus sativus uses to produce Safranal, Crocin, and Picrocrocin. E. Coli was also an option because we had found 2 constructs from the parts registry that we could use to make E. Coli produce Zeaxanthin.
 
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One of the first decisions we had to make was to pick which organism we wanted to work with. It was a difficult decision because each had their own positives and negatives. E. Coli was easy to clone, fast growing and we could measure our products using spectroscopy. However since our goal is to produce a compound that people will eventually use in their food, we were hesitant because of the public perception of E. Coli. Synechocystis PCC 6803 was naturally competent, it naturally produces zeaxanthin, however it has a slow growth cycle and you cannot measure our products with spectroscopy. We ultimately decided to use both E. Coli and Synechosystis.  
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<area shape="rect" coords="70,75,90,125" href="http://partsregistry.org/Help:BioBrick_Prefix_and_Suffix" />
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<area shape="rect" coords="100,75,210,125" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K906003" />
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<area shape="rect" coords="350,75,460,125" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K906003" />
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<area shape="rect" coords="330,75,350,125" href="http://partsregistry.org/Help:BioBrick_Prefix_and_Suffix" />
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<area shape="rect" coords="588,75,610,125" href="http://partsregistry.org/Help:BioBrick_Prefix_and_Suffix" />
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<area shape="rect" coords="235,75,330,125" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K906000" />
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<area shape="rect" coords="480,75,580,125"href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K906002" />
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<area shape="rect" coords="370,210,690,260" href="http://partsregistry.org/Part:pSB1C3" />
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<area shape="rect" coords="0,75,0,125"href="http://partsregistry.org/Help:BioBrick_Prefix_and_Suffix" />
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</map>
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==Rising Action==
 
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==Climax==
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</body>
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</html>
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<br><br>
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==Falling Action==
 
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==Dénouement==
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<h3>Results</h3>
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The soluble protein in the protein which was transformed with the tagged pro was extracted and analyzed by SDS-PAGE gel electrophoresis.  The result showed showed a protein of the target mass that was found primarily in the induced cells.  This result was further confirmed by digestion of the band in question using trypsin (Sigma Aldrich, St. Louis) and analyzed using LC-MS (Waters Synapt G2 and HPLC).  <br>
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<img src="https://static.igem.org/mediawiki/2012/5/5f/LCMS_gel.png" width="200">
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<img src="https://static.igem.org/mediawiki/2012/a/a7/Zcd_coveage.png" width="500"><br>
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<img src="https://static.igem.org/mediawiki/2012/d/d9/Screen_Shot_2012-09-14_at_10.46.37_PM.png" width="700">
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<br>
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The information that lead to the development of the this vector tag with ZCD came out of the following paper:
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Bouvier, Florence et al. “Oxidative Remodeling of Chromoplast Carotenoids : Identification of the Carotenoid Dioxygenase CsCCD and CsZCD Genes Involved in Crocus Secondary Metabolite Biogenesis.” 15.January (2003): 47–62.
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</html>

Latest revision as of 00:01, 2 October 2012




Phase II

Design

The purpose of phase II is to optimize the expression of the proteins necessary for the production of crocin glycosides and safranal. Unfortunately, ZCD has been shown to produce inclusion bodies when expressed'' in vivo'' in ''E. coli'' and so an alternative method needed to be developed to create a functional protein. This was done by following the technique described by Florence Bouvier (The Plant Cell, vol. 15 47-62).


Results

The soluble protein in the protein which was transformed with the tagged pro was extracted and analyzed by SDS-PAGE gel electrophoresis. The result showed showed a protein of the target mass that was found primarily in the induced cells. This result was further confirmed by digestion of the band in question using trypsin (Sigma Aldrich, St. Louis) and analyzed using LC-MS (Waters Synapt G2 and HPLC).


The information that lead to the development of the this vector tag with ZCD came out of the following paper: Bouvier, Florence et al. “Oxidative Remodeling of Chromoplast Carotenoids : Identification of the Carotenoid Dioxygenase CsCCD and CsZCD Genes Involved in Crocus Secondary Metabolite Biogenesis.” 15.January (2003): 47–62.

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