"
Team:Uppsala/astaxanthin
From 2013.igem.org
Astaxanthin
Astaxanthin is a carotenoid in the carotenoid pathway that derives from the xanthophyll group and the precursor canthaxanthin is catalyzed by CrtZ. Astaxanthin is a very strong antioxidant but studies have shown that it also has other health benefits. The substance is created by many marine bacteria and microalgae and it gives the characteristic red color to many species such as salmon, lobster and shrimps.
Astaxanthin is an expensive part of salmon farming, used to give the meat its red color and it is approved in many countries as a food coloring with the e-number 161.[1] When astaxanthin is produced it is usually by using the microalgae Haematococcus, or by producing it synthetically. The costs for astaxanthin today is around 2500 US dollars per kg, in total the market is worth around 200 million US dollars a year.[2] Since astaxanthin has many health benefits and that there is a demand for it on the market we decided to do more work on it to make it more available.
Methods
To see that the bacteria containing the operon expressed astaxanthin we had to characterize them. To do that we used a spectrophotometer (nanodrop 2000 UV/Vis spectrophotometer) and we extracted astaxanthin from salmon as a standard. After growing bacteria cultures with the operon we later used different extraction methods to extract the astaxanthin with various results.
We got the operon for producing astaxanthin from the Slovenian iGEM team 2010 which consisted of the genes CrtE, CrtB, CrtI, CrtY, CrtO and CrtZ with RBS, linkers, zinc fingers and the inducible promoter pBAD/AraC. We inserted the operon in e-coli DH5alpha. The zinc fingers aren´t of much interest to us so we won´t use them for any other parts.
Results
To see that the bacteria containing the operon expressed Astaxanthin we had to characterize them. To do that we used a spectrophotometer (nanodrop 2000 UV/Vis spectrophotometer) and we extracted Astaxanthin from salmon as a standard. after growing bacteria cultures with the operon we later used different extraction methods to extract the Astaxanthin with various results.
Figure 1, mixed salmon that we boiled in methanol and centrifuged.
Figure 2, liquid liquid extraction where methanol was used as the eluent
In the end we may have gotten a successful result but the extraction method was not good enough to get a clear graph proving the existence of astaxanthin, the extraction could only indicate that there is a possibility that we might have astaxanthin. See graph
Spectrophotometry
 | Figure 3 Our standard for astaxanthin from salmon, the peak in general is at the correct wavelength although it´s not the strongest, probably due to low concentration in the solution. |
 | Figure 4 Negative Control in methanol. |
 | Figure 5 We did a few extraction attempts for astaxanthin but we didn´t manage to get a clean graph of astaxanthin, although we did get indication of the existence with most of the different tries. The graph above is one try that have the focus around the correct point but there are too many other thing to prove its existence. |
