Team:Uppsala/zeaxantin

From 2013.igem.org

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<h1>β-carotene</h1>
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<h1>Zeaxanthin</h1>
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<p>β-carotene is a common carotenoid that derives from the xanthophyll group known for its characteristic orange color. β-carotene can be found in fruits and vegetables, for example carrots. β-carotene is a provitamin to vitamin A, which means that during the right circumstances β-carotene can be catalyzed further to produce vitamin A. Vitamin A has several important health aspects which includes skin, mucus membrane and most common to people a good effect on eyes and vision. Studies have shown that the carotenoid can prevent aging of the brain and stimulate it to keep its capability of memory when a high ascorbic acid concentration also is present <sup><a href="#l1">(1)</a></sup>. The health aspects were an important part of our choice to produce β-carotene.</p>
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<p>Zeaxanthin is a yellow carotenoid pigment, derived from the precursor ß-carotene through hydroxylation by the enzyme ”Beta-carotene hydroxylase”. Zeaxanthin acts as an antioxidant and can be found in for example peppers, yolk and maize.<sup><a href="#l1">(1)</a></sup> According to studies zeaxanthin has positive effects on both undamaged and impaired vision and it may prevent age-related macular degeneration (AMD), an eye condition that could lead to blindness. Furthermore studies have also indicated that zeaxanthin may have skin protective activities<sup><a href="#l2"> (1,4).</a></sup>
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<br><br>
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<h1>Methods</h1>
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Except for having beneficial properties the carotenoid the carotenoid zeaxanthin is one of the primary precursors for the production of the <a href="https://2013.igem.org/Team:Uppsala/saffron">saffron metabolites</a> picrocrocin, crocin and safranal. The production of saffron was inspired by the work of Washington team <a href="https://2012.igem.org/Team:WashU">WashU iGEM 2012.</a> Since the gene (CrtZ) that was used for the production of zeaxanthine was a eukaryotic version from Arabidopsis Thaliana, we decided to focus on finding a suitable gene to optimize the production of the above-mentioned metabolites. When searching for an appropriate gene we came in contact with Slovenia iGEM 2010 (lank) that had produced Zeaxanthin in E.coli. The operon that was obtained contained genes that were originally from the bacteria <a href="http://parts.igem.org/Part:BBa_K323122">Pantoea Anantis.</a></p>  
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<p>The gene CrtY that is responsible for translating the protein lycopene cyclase was obtained from the zeaxanthin operon provided by Slovenia iGEM team 2010. Production of β-carotene is initiated through the catalytic reaction done by the enzyme lycopene cyclase which uses the precursor <a href="https://2013.igem.org/Team:Uppsala/lycopene">lycopene.</a></p>
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<h1>Methods:</h1>
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<h1>Results (Summary)</h1>
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<p>For the characterization of zeaxanthin in E.coli DH5alpha we put together a standardized method for zeaxanthin extraction and measuring. Since zeaxanthin in its pure form is extremely expensive (~ 480 €/mg) we bought maize at our local supermarket and carried out liquid-liquid extractions using methanol and performed spectrophotometry measurements. We compared literature absorbance values of zeaxanthin to the peaks we obtained from our own measurements and continued our experiments until we believed to have an optimized extraction method.</p>
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<p>Beta carotene production was proven through the production of our operon containing <a href="https://2013.igem.org/Team:Uppsala/zeaxantin">zeaxanthin.</a> Since production of zeaxanthin relies on beta carotene as a precursor we could simultaneously prove that we successfully had produced both zeaxanthin and beta carotene through liquid-liquid separation and spectrophotometry <a href="https://2013.igem.org/Team:Uppsala/zeaxantin">(read spectrophotometry of zeaxanthin for more results)</a></p>
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<img class="zeaxanthin1" src="">
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<img class="zeaxanthin2" src="">
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<i>Figure 1. Maize bought at our local supermarket that was grinded and later used to make a standardized zeaxanthin extraction method </i>
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<i>Figure 2. Liquid-liquid extraction experiments carried out on grinded maize using methanol.</i>
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<p>The operon acquired from Slovenia iGEM 2010 consists of the genes CrtE, CrtB, CrtI, CrtY and CrtZ connected through zink fingers and linkers and assembled with the inducible promoter pBAD/AraC. In our project the zink fingers were not put into a lot of consideration.
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Since we would eventually like to produce zeaxanthin in Lactobacillus in yoghurt it would be preferable to have a constitutive promoter, such as the CP-promoters. Parallel to the characterization of zeaxanthin in E.coli we therefore attempted to remove the pBAD/AraC promoter from the zeaxanthin operon.</p>
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<h1>Results:</h1>
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<h1 class="no-border">Spectrophotometry</h1>
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<p>Production of zeaxanthin in e-coli DH5alpha was detected trough spectrophotometry measurements performed after liquid-liquid extractions using methanol. The resulting absorbance curve was compared to the standardized curve. In both graphs you can find the two peaks corresponding to zeaxanthin.</p>
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<img class="zeaxanthin3" src="">
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<img class="zeaxanthin4" src="">
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<i>Figure 3. E.coli culture believed to have zeaxanthin production</i>
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<i>Figure 4. A liquid-liquid extraction was carried out on the E.coli culture containing the plasmid above. Methanol was used as an organic solvent.</i>
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<img class="zeaxanthin5" src="">
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<img class="zeaxanthin6" src="">
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<i>Figure 5. Spectrophotometry of zeaxanthin extraction from maize resulted in the graph above. The two peaks are characteristic for zeaxanthin.</i>
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<i>Figure 6. Spectrophotometry of an E.coli culture containing the plasmid with the zeaxanthin operon. The two characteristic peaks of zeaxanthin are present, indicating that zeaxanthin was present in the sample.</i>
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<p>Spectrophotometry measurements were also done on unmodified E.coli, as a negative control. These measurements did not result in the peaks that are characteristic for zeaxanthin. </p>
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<img class="zeaxanthin7" src="">
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<i>Figure 7. Spectrophotometry of unmodified E.coli did not result in the two peaks characteristic for zeaxanthin. </i>
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<h1>Results (Bio-bricks)</h1>
 
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<p>We realized that there were a lot of components in the carotenoid pathway that we were in need of in the beginning of the project that were missing in the registry. Therefore after obtaining the zeaxanthin operon from Slovenias iGEM team 2010 we directly proceeded with isolating of the genes as basic components in order to send them to the registry. </p>
 
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Revision as of 02:52, 28 September 2013

Uppsala iGEM 2013

Zeaxanthin

Zeaxanthin is a yellow carotenoid pigment, derived from the precursor ß-carotene through hydroxylation by the enzyme ”Beta-carotene hydroxylase”. Zeaxanthin acts as an antioxidant and can be found in for example peppers, yolk and maize.(1) According to studies zeaxanthin has positive effects on both undamaged and impaired vision and it may prevent age-related macular degeneration (AMD), an eye condition that could lead to blindness. Furthermore studies have also indicated that zeaxanthin may have skin protective activities (1,4).

Except for having beneficial properties the carotenoid the carotenoid zeaxanthin is one of the primary precursors for the production of the saffron metabolites picrocrocin, crocin and safranal. The production of saffron was inspired by the work of Washington team WashU iGEM 2012. Since the gene (CrtZ) that was used for the production of zeaxanthine was a eukaryotic version from Arabidopsis Thaliana, we decided to focus on finding a suitable gene to optimize the production of the above-mentioned metabolites. When searching for an appropriate gene we came in contact with Slovenia iGEM 2010 (lank) that had produced Zeaxanthin in E.coli. The operon that was obtained contained genes that were originally from the bacteria Pantoea Anantis.

Methods:

For the characterization of zeaxanthin in E.coli DH5alpha we put together a standardized method for zeaxanthin extraction and measuring. Since zeaxanthin in its pure form is extremely expensive (~ 480 €/mg) we bought maize at our local supermarket and carried out liquid-liquid extractions using methanol and performed spectrophotometry measurements. We compared literature absorbance values of zeaxanthin to the peaks we obtained from our own measurements and continued our experiments until we believed to have an optimized extraction method.

Figure 1. Maize bought at our local supermarket that was grinded and later used to make a standardized zeaxanthin extraction method Figure 2. Liquid-liquid extraction experiments carried out on grinded maize using methanol.

The operon acquired from Slovenia iGEM 2010 consists of the genes CrtE, CrtB, CrtI, CrtY and CrtZ connected through zink fingers and linkers and assembled with the inducible promoter pBAD/AraC. In our project the zink fingers were not put into a lot of consideration.

Since we would eventually like to produce zeaxanthin in Lactobacillus in yoghurt it would be preferable to have a constitutive promoter, such as the CP-promoters. Parallel to the characterization of zeaxanthin in E.coli we therefore attempted to remove the pBAD/AraC promoter from the zeaxanthin operon.

Results:

Spectrophotometry

Production of zeaxanthin in e-coli DH5alpha was detected trough spectrophotometry measurements performed after liquid-liquid extractions using methanol. The resulting absorbance curve was compared to the standardized curve. In both graphs you can find the two peaks corresponding to zeaxanthin.

Figure 3. E.coli culture believed to have zeaxanthin production Figure 4. A liquid-liquid extraction was carried out on the E.coli culture containing the plasmid above. Methanol was used as an organic solvent. Figure 5. Spectrophotometry of zeaxanthin extraction from maize resulted in the graph above. The two peaks are characteristic for zeaxanthin. Figure 6. Spectrophotometry of an E.coli culture containing the plasmid with the zeaxanthin operon. The two characteristic peaks of zeaxanthin are present, indicating that zeaxanthin was present in the sample.

Spectrophotometry measurements were also done on unmodified E.coli, as a negative control. These measurements did not result in the peaks that are characteristic for zeaxanthin.

Figure 7. Spectrophotometry of unmodified E.coli did not result in the two peaks characteristic for zeaxanthin.

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