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| | <h3>Results</h3> | | <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> |
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| - | <img src="https://static.igem.org/mediawiki/2012/5/5f/LCMS_gel.png" width="300"> | + | <img src="https://static.igem.org/mediawiki/2012/5/5f/LCMS_gel.png" width="200"> |
| - | <img src="https://static.igem.org/mediawiki/2012/a/a7/Zcd_coveage.png" width="500"> | + | <img src="https://static.igem.org/mediawiki/2012/a/a7/Zcd_coveage.png" width="500"><br> |
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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>[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>
| + | The information that lead to the development of the this vector tag with ZCD came out of the following paper: |
| - | 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.
| + | 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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| - | 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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| - | <td><a href="/Team:WashU/Saffroninakansynecho"><img src ="http://aux4.iconpedia.net/uploads/937908040.png" width=82px height=82px></a></td>
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