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| | __NOTOC__ | | __NOTOC__ |
| - | {{WashUback}} | + | {{WashUbackSaffron}} |
| | {{WashUprojectbar}} | | {{WashUprojectbar}} |
| | <html> | | <html> |
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| | <div id = "design"> | | <div id = "design"> |
| - | <div align ="center"><font size="5"><u><b>Our Design</b></u></font></div> | + | <h1>Phase II</h1> |
| - | <br><br>
| + | <h3>Design</h3> |
| - | <div align ="center"><font size="4"><b>The Gene</b></font></div><br><br>
| + | 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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| | <body> | | <body> |
| - | <IMG STYLE="position:absolute; TOP:470px; LEFT:170px" img src="https://static.igem.org/mediawiki/igem.org/2/2f/Thiofusion_diagram_2.jpg" ALIGN=RIGHT width="786" border="0" usemap="#map" > | + | <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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| | <!-- #$VERSION:2.3 --> | | <!-- #$VERSION:2.3 --> |
| | <!-- #$AUTHOR:tony --> | | <!-- #$AUTHOR:tony --> |
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| - | <area shape="rect" coords="79,89,147,129" href="blank.org" /> | + | <area shape="rect" coords="100,75,210,125" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K906003" /> |
| - | <area shape="rect" coords="157,88,228,133" href="http://www.ncbi.nlm.nih.gov/protein/75146812?report=genbank&amp;amp;log$=prottop&amp;amp;blast_rank=1&amp;amp;RID=Y1R3SV1R01S" /> | + | <area shape="rect" coords="350,75,460,125" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K906003" /> |
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| - | <area shape="rect" coords="313,88,381,132" href="http://www.ncbi.nlm.nih.gov/protein/33114570?report=genbank&amp;amp;log$=prottop&amp;amp;blast_rank=1&amp;amp;RID=Y1RGDZF001S" /> | + | <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="471,89,534,133" href="http://www.ncbi.nlm.nih.gov/protein/15235959" /> | + | <area shape="rect" coords="480,75,580,125"href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K906002" /> |
| - | <area shape="rect" coords="556,85,614,133" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_B1002" /> | + | <area shape="rect" coords="370,210,690,260" href="http://partsregistry.org/Part:pSB1C3" /> |
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| | </map> | | </map> |
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| | </body> | | </body> |
| | </html> | | </html> |
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| - | <br><br><br><br><br>
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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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| | <br><br> | | <br><br> |
| - | 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>
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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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| - | <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 zeaxanthin produced to drive the synthesis to the products. We feared that inherent, natural back-regulation could slow the endogenous production of zeaxanthin even if we could find ideal conditions for zeaxanthin production. Therefore, we added a non-native gene that will constitutively produce zeaxanthin even in the event of back-regulation. Since this gene is more of a bonus producer than a necessity, it was last since a the other two genes were more important.
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| - | <br>
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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. In between the cut sites is an "ATA" spacer sequence to help ensure efficiency and fidelity in the digests. <br>
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| - | Terminator<br>
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| - | We chose a small, efficient (85%) terminator from 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 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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| | + | <h3>Results</h3> |
| | + | 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> |
| | <html> | | <html> |
| - | | + | <img src="https://static.igem.org/mediawiki/2012/5/5f/LCMS_gel.png" width="200"> |
| - | <body> | + | <img src="https://static.igem.org/mediawiki/2012/a/a7/Zcd_coveage.png" width="500"><br> |
| - | <div align = "center">
| + | <img src="https://static.igem.org/mediawiki/2012/d/d9/Screen_Shot_2012-09-14_at_10.46.37_PM.png" width="700"> |
| - | <table>
| + | <br> |
| - | <table id="arrowtable">
| + | The information that lead to the development of the this vector tag with ZCD came out of the following paper: |
| - | <style type="text/css">
| + | 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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| - | </style>
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| - | <tr>
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| - | <td><a href="/Team:WashU/Saffroninakansynecho"><img src =" http:// aux4. iconpedia. net/ uploads/ 937908040.png" width= 82px height=82px>< /a></td> | + | |
| - | <td><a href="/Team:WashU/#"><img src =" http:// aux. iconpedia. net/ uploads/ 1782679686.png" width= 82px height=82px></a></td> | + | |
| - | </tr> | + | |
| - | </table>
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| - | </div>
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| - | </body>
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| | </html> | | </html> |
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