Team:WashU/DesignSynecho

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<div align ="center"><font size="5"><u><b>Our Design</b></u></font></div>
 
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<div align ="center"><font size="4"><b>The Gene</b></font></div>
 
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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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==Gene Design==
 
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BioBrick Prefix/Suffix<br>
 
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The Biobrick prefix and suffix was added to our gene so that they could be biobricked on to any biobrick plasmid with ease.
 
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Constitutive promoter with RBS<br>
 
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Bert Berla, a graduate student adviser, helped us chose this promoter. It works well with synechocystis and contains a downstream RBS region that we use for our RBS regions.
 
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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 (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 from crocin dialdehyde. Picrocin naturally degrades to saffranal. Thus this enzyme finishes the production of our desired compounds.
 
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<br>[http://www.ncbi.nlm.nih.gov/protein/15235959?report=genbank&log$=prottop&blast_rank=1&RID=Y1RMH11X01S CrtZ]. <br>
 
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CrtZ (β-carotene hydroxylase) makes zeaxanthin from β-carotene. We wanted to include this gene even though ''Synechocystis'' produces Zeaxanthin endogenously because we wanted to increase the amount of endogenously produced zeaxanthin produced so that we could produce more product.
 
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<br>RBS, Restriction sites<br>
 
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In between each gene we added Ribosome binding sites and restriction sites. The ribsome binding sites were necessary for the expression of our construct. The Restriction sites were added so that the genes could be easily cut out of the construct and manipulated. <br>
 
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Terminator<br>
 
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Our terminator was just a regular terminator we found on the parts registry.<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 synthesize.
 
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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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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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