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<div class="miniheader"> Activity measurements of purified CsADH2946 </div> | <div class="miniheader"> Activity measurements of purified CsADH2946 </div> | ||
<div> To verify the activity of our purified enzyme CsADH2946 to convert crocetin dialdehyde to crocetin, an activity measurements(link protocol) assay was performed on a plate reader measuring absorbance of the substrate and product of the reaction. For the experiment we used a 96-well plate in which we included wells with enzyme from pooled fractions + substrate, as well as positive and negative controls, see table Z for the specifics. </div> | <div> To verify the activity of our purified enzyme CsADH2946 to convert crocetin dialdehyde to crocetin, an activity measurements(link protocol) assay was performed on a plate reader measuring absorbance of the substrate and product of the reaction. For the experiment we used a 96-well plate in which we included wells with enzyme from pooled fractions + substrate, as well as positive and negative controls, see table Z for the specifics. </div> | ||
+ | |||
+ | <figure class="figure"> | ||
+ | <figcaption class="figure-caption" style="padding-left: 20%; padding-right: 20%"> Table Z. Content of wells used for activity measurement of CsADH2946.</figcaption> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/7/7c/CraftingCrocinTableStep2.png" class="figure-img img-fluid" style="display: block; margin: auto; width: 40%; height: auto; padding-top: 2%; padding-bottom: 5%;"> | ||
+ | </figure> | ||
+ | |||
<div> As can be seen in figure X, the absorbance of the product crocetin increases over time in well 2 containing enzyme and the substrate crocetin dialdehyde. After 9 hours of reaction, the blue curve corresponding to the enzyme + substrate mixture has increased its absorbance in the exact range of the product. The negative and positive control curves look similar to time point zero, apart from some precipitation of product and substrate indicated by the decreased curves. A definite evidence that we succeeded to produce a functional CsADH2946 enzyme. </div> | <div> As can be seen in figure X, the absorbance of the product crocetin increases over time in well 2 containing enzyme and the substrate crocetin dialdehyde. After 9 hours of reaction, the blue curve corresponding to the enzyme + substrate mixture has increased its absorbance in the exact range of the product. The negative and positive control curves look similar to time point zero, apart from some precipitation of product and substrate indicated by the decreased curves. A definite evidence that we succeeded to produce a functional CsADH2946 enzyme. </div> | ||
+ | |||
<div> In addition, in figure X we can see that well 2 containing enzyme and crocetin dialdehyde has changed color compared to the negative control, to become more yellow like the product crocetin in well 8. This also shows that CsADH2946 was produced and that it converts crocetin dialdehyde into crocetin. | <div> In addition, in figure X we can see that well 2 containing enzyme and crocetin dialdehyde has changed color compared to the negative control, to become more yellow like the product crocetin in well 8. This also shows that CsADH2946 was produced and that it converts crocetin dialdehyde into crocetin. | ||
+ | </div> | ||
+ | <figure class="figure"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/d/d3/CraftingCrocinActivityStep2.png" class="figure-img img-fluid" style="display: block; margin: auto; width: 40%; height: auto; padding-top: 5%; padding-bottom: 2%;"> | ||
+ | <figcaption class="figure-caption" style="padding-left: 20%; padding-right: 20%"> Figure X. Activity measurement curve. The dotted lines describe wells at the starting time and the fully drawn lines describe the absorbance after 9 hours. The blue lines indicate wells containing protein and crocetin dialdehyde, red lines describe positive control with only crocetin dialdehyde and black lines correspond to the negative control only containing the desired product crocetin.</figcaption> | ||
+ | </figure> | ||
+ | <figure class="figure"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/b/bf/CraftingCrocinWellStep2.png" class="figure-img img-fluid" style="display: block; margin: auto; width: 40%; height: auto; padding-top: 5%; padding-bottom: 2%;"> | ||
+ | <figcaption class="figure-caption" style="padding-left: 20%; padding-right: 20%"> Figure X. 96-well plate for activity measurements post 24 hours reaction and plate reading. The plate include pooled enzyme fractions 10-15 + substrate crocetin dialdehyde (well 1), pooled enzyme fractions 16-23 + crocetin dialdehyde (well 2), flow through + crocetin dialdehyde (well 3), negative control with only crocetin dialdehyde (well 4), pooled enzyme fractions 10-15 + product crocetin (well 5) pooled enzyme fractions 16-23 + crocetin (well 6), flow through + crocetin (well 7) and positive control with only crocetin (well 8).</figcaption> | ||
+ | </figure> | ||
+ | <div class="miniheader"> Modeling of CsADH2946 </div> | ||
+ | <div> Since the enzyme is poorly characterized, we created a homology model and performed stability simulations to verify that our model was reasonable. The homology modeling revealed that CsADH2946 is in fact tetrameric, which helped us in the purification and characterization process. We performed a pulling simulation between the enzyme and its substrate in order to estimate binding energy and calculate a theoretical KM. The resulting structure of the homology modeling and a plot of the pulling simulation can be seen in figure X. Read more about the homology modeling and dynamics modeling in the Modeling section. | ||
+ | </div> | ||
+ | <figure class="figure"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/d/df/CraftingCrocinModelingStep2.png" class="figure-img img-fluid" style="display: block; margin: auto; width: 40%; height: auto; padding-top: 5%; padding-bottom: 2%;"> | ||
+ | <figcaption class="figure-caption" style="padding-left: 20%; padding-right: 20%"> Figure X. Homology model of CsADH2946 and a plot demonstrating the pulling of the substrate crocetin dialdehyde from the active site of CsADH2946. | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <div class="miniheader"> Estimation of KM </div> | ||
+ | <div> Since the enzyme is poorly characterized, we created a homology model and performed stability simulations to verify that our model was reasonable. The homology modeling revealed that CsADH2946 is in fact tetrameric, which helped us in the purification and characterization process. We performed a pulling simulation between the enzyme and its substrate in order to estimate binding energy and calculate a theoretical KM. The resulting structure of the homology modeling and a plot of the pulling simulation can be seen in figure X. Read more about the homology modeling and dynamics modeling in the Modeling section. | ||
</div> | </div> | ||
Revision as of 21:20, 30 October 2017
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We at Uppsala this year, are planning to make Alpha crocin in E.coli. Alpha-crocin, an apocarotenoid found in Crocus and Gardenia, is responsible for the red color of Saffron. Recent studies suggest that crocin may have several medicinal properties.Due to its colour, it could also be potentially used as a dye. It being a powerful antioxidant with interesting and not yet fully studied medicinal capabilities, large scale mass production of crocin would be of interest to further study its effects on the human body. Our team from 2013 already did the groundwork for us by developing zeaxanthin accumulating strain of E.coli. This year's project is building up on that. We identified three enzymatic steps leading from zeaxanthin to crocin.