Team:WashU/DesignSynecho

Gene Design

BioBrick Prefix/Suffix The Biobrick prefix and suffix was added to our gene so that they could be biobricked on to any biobrick plasmid with ease.

Constitutive promoter with RBS Bert Berla, a graduate student adviser, helped us chose this promoter. It works well with synechocystis and contains a downstream RBS region that we use for our RBS regions.

[http://www.ncbi.nlm.nih.gov/protein/75146812?report=genbank&log$=prottop&blast_rank=1&RID=Y1R3SV1R01S ZCD] This gene is from the organism Crocus sativus. We decided on this gene because it was derived from a plant and so would be better expressed in our model organism. We put this gene first because it is the first enzyme to cleave zeaxanthin to crocin and safranal precursors. Since Crtz is endogenous in Synechocystis we decided to put this gene last.

[http://www.ncbi.nlm.nih.gov/protein/33114570?report=genbank&log$=prottop&blast_rank=1&RID=Y1RGDZF001S UGTCS2] This gene is also from Crocus sativus. We chose it for a similar reason to that of ZCD.

[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 increase the amount of endogenously produced zeaxanthin produced so that we could produce more product.

RBS, Restriction sites 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.

Terminator 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.