Overview Phase I Phase II Phase III Results Parts Judging Criteria
The first thing we did was figuring out the carotenoid biochemical pathway from beta carotene to our desired products safranal, crocin and picrocrocin. We noticed that the pathway was very complex and lengthy. Therefore we wanted to simplify things by finding an organism that endogenously produced one of the intermediates close to our final products. Our advisers suggested using Synechocystis PCC 6803, which produced beta carotene and many of its derivatives including Zeaxanthin which Crocus sativus uses to produce Safranal, Crocin, and Picrocrocin. E. Coli was also an option because we had found 2 constructs from the parts registry that we could use to make E. Coli produce Zeaxanthin.
One of the first decisions we had to make was to pick which organism we wanted to work with. It was a difficult decision because each had their own positives and negatives. E. Coli was easy to clone, fast growing and we could measure our products using spectroscopy. However since our goal is to produce a compound that people will eventually use in their food, we were hesitant because of the public perception of E. Coli. Synechocystis PCC 6803 was naturally competent, it naturally produces zeaxanthin, however it has a slow growth cycle and you cannot measure our products with spectroscopy. We ultimately decided to use both E. Coli and Synechosystis.
We started to work on the gene we were going to transform E. Coli and Synechocystics early. We decided to construct 3 genes and put them in a plasmid that could homogeneously recombine in Synechocystis. The 3 genes we chose were ZCD, UGTCS2 and CrtZ. CrtZ (β-carotene hydroxylase) makes zeaxanthin from β-carotene. We wanted to include this gene even though Synechocystis produces Zeaxanthin endogenously because we wanted to make sure increase the amount of endogenously produced zeaxanthin produced so that we could produce more product. ZCD and UGTCS2 were the two enzymes that cleaved zeaxanthin to make our products. We picked a promoter that our advisers suggested because it had been shown in the Pakrasi lab ( a lab at WashU that works with Synechocystis) to work well in Synechocystis. In between each gene we added Ribosome binding sites and restriction sites. The ribsome binding sites were necessary for the expression of our construct. The Restriction sites were added so that the genes could be easily cut out of the construct and manipulated. Our terminator was just a regular terminator we found on the parts registry. We optimized the construct for Synechocystis PCC 6803 using a program from DNA 2.0. We submitted the gene to DNA 2.0 to synthesize.
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