Parts

Our team this year focused on producing a natural occurring dye. We found the chromoproteins from the 2013 Uppsala iGEM team and decided to pursue chromoproteins as an alternative to the environmentally damaging azo dyes.

From last year’s project, the team was already familiar with the CIDAR MoClo Kit and its many options for building tailor made expression vectors. These essential parts like the constitutive promoters, ribosome binding sites, terminators, and backbones were combined with sequences we designed and synthesized to construct dye producing E. coli.

Listed below are a number of gene fragments ((6)-(20)) that we adapted and combined from existing iGEM parts to fit the MoClo Golden Gate cloning scheme. We designed these sequences with the meffRed chromoprotein and two different cellulose binding domains in two orientations. Our constructs with meffRed later proved to show only faint color so we transitioned to the mScarlet fluorescent protein which is much brighter than meffRed even under normal light. We also adapted an arabinose induced promoter to fit our cloning scheme. Parts were codon optimized for E. coli and designed to be Golden Gate compatible with Bsa1 sites for cloning into a circular plasmids for dye production as well as Bpi1 sites for cloning the parts into the pOGG006 backbone for long-term storage in a bacterial stock.

Our project after designing the gene fragments involved cloning these parts together and characterizing the plasmid products as expression vectors for cotton fabric dyes. The rest of the parts listed in the table below are those expression vectors. We went through many unsuccessful combinations and iterations before finding a construct that produced a lot of bright dye while still binding to cellulose (cotton). pGEC046 (BBa_K4258046) is the culmination of our work this year.