Difference between revisions of "Team:Uppsala/AlphaCrocin"

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. 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.

We successfully made a sequence verified BioBrick of CaCCD2 with His-tag. The BioBrick was also combined with the other steps in the pathway and inserted into the zeaxanthin producing E. coli strain for a complete pathway from FPP to crocin. See the result here!

Since the enzyme is poorly characterized, we created a homology model and performed stability simulations to verify that our model was reasonable. The resulting structure of the homology modelling and its corresponding RMSD plot from simulations can be seen in figure X. The RMSD plot indicated that the model conforms to a stable structure. Read more about the homology modelling and dynamics modelling in the Modelling section.

We are the first to express and characterize CsADH2946! This aldehyde dehydrogenase gene from Crocus Sativus has previously only been identified as a candidate gene through proteome analysis, and has thus never been isolated or characterized before [2]. We successfully made a sequence verified BioBrick of CsADH2946 with His-tag. The BioBrick was also combined with the other steps in the pathway and inserted into the zeaxanthin producing E. coli strain for a complete pathway from FPP to crocin. See the result here!

CsADH2946 was transformed and expressed in E. coli strain BL21 (DE3*) and purified using IMAC(link protocol) on an ÄKTA protein purification system. We used a gradient of imidazole concentration from 20-500 mM, in order to get our enzyme as separated as possible from other proteins that ends up in the fractions. The peak pointed at by the arrow in the chromatogram (Figure X) indicates protein that elutes at high imidazole concentration, i.e our desired His-tagged CsADH2946. The purification was followed by SDS-PAGE to analyse the fractions, control purity and verify the protein product. In figure X the band at around 60 kDa in the crude pellet indicate an overexpression of a protein in that size range. In the SDS gel of fractions 16-26 collected between 115 - 145 mL elution volume there is a strong band at 60 kDa corresponding to the molecular weight of CsADH2946, indicating that our protein was successfully overexpressed and well-separated.

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.

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.

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.