The Gene

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Gene Design

We decided to construct 3 genes and put them in a plasmid that could homogeneously recombine in Synechocystis. The 3 genes we chose were [http://www.ncbi.nlm.nih.gov/protein/75146812?report=genbank&log$=prottop&blast_rank=1&RID=Y1R3SV1R01S ZCD], [http://www.ncbi.nlm.nih.gov/protein/33114570?report=genbank&log$=prottop&blast_rank=1&RID=Y1RGDZF001S UGTCS2] and [http://www.ncbi.nlm.nih.gov/protein/15235959?report=genbank&log$=prottop&blast_rank=1&RID=Y1RMH11X01S 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 (Zeaxanthin 7,8-dioxygenase) and UGTCS2 (Glucosyltransferase 2) were the two enzymes that cleaved zeaxanthin to make our products. 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.