Team:WashU/DesignSynecho

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<h1>Phase III</h1>
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<h1>Phase II</h1>
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The purpose of Phase III is to complete the production of safranal and crocin by putting together the biobricks from Phase II.
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<h3>Design</h3>
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<img src="http://dl.dropbox.com/u/88390549/CrocinGraphs2.png"> <br><br>
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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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==Gene Design==
 
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BioBrick Prefix/Suffix<br>
 
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The Biobrick prefix and suffix were added to our gene so that they could be biobricked onto any biobrick plasmid with ease.
 
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<br><br>
 
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<br>[http://www.ncbi.nlm.nih.gov/protein/75146812?report=genbank&log$=prottop&blast_rank=1&RID=Y1R3SV1R01S ZCD]<br>
 
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This gene is from the organism ''Crocus sativus''. This gene is only found in the Crocus species that produce this unique spice naturally. Thus, this gene is certainly necessary. We put this gene first because it is the first enzyme to cleave zeaxanthin to crocin dialdehyde, the precursor to crocin glycosides (the primary pigment of Saffron), and hydroxy-beta-cyclocitral, the precursor to picrocin and safranal (a significant portion of the aroma of saffron). Since CrtZ has an endogenous ortholog in ''Synechocystis'', CrtR, we decided to put this gene last.
 
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<br>[http://www.ncbi.nlm.nih.gov/protein/33114570?report=genbank&log$=prottop&blast_rank=1&RID=Y1RGDZF001S UGTCS2]<br>
 
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This gene is also from ''Crocus sativus''. We chose it for a similar reason to that of ZCD. This gene produces picrocin from hydroxy-beta-cyclocitral and crocin glycosides from crocin dialdehyde. Picrocin naturally degrades to saffranal. Thus, this enzyme finishes the production of our desired compounds.
 
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Terminator<br>
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<h3>Results</h3>
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We chose a small, efficient (85%) terminator from the parts registry.<br>
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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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We optimized the construct for ''Synechocystis'' PCC 6803 using a program from DNA 2.0. We submitted the gene to DNA 2.0 to further evaluate and synthesize. DNA 2.0 provided the service of analyzing secondary structure of the gene near the RBS to help ensure successful translation.
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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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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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