Dyenamix: A Better Alternative to Azo Dyes
Our team this year focused on producing a biosynthetic dye to replace traditional, harmful textile dyes which can be dangerous to both humans and natural ecosystems. They may contain carcinogens, heavy metals, toxins, and photosynthesis reducing agents. In addition, synthetic azo dyes are highly stable in aquatic ecosystems, making them hard to remove. To address this problem, our team is using synthetic biology to create a protein based red dye, specifically engineered to bind with cotton.
Chromoproteins were an attractive option for the foundation of the project as their expression in Escherichia coli has been well characterized. They were therefore a great choice for us to produce the basis for a better alternative to azo dyes within a limited time. Our project first started with a single chromoprotein, meffRed, which came from the 2013 Uppsala team on the iGEM parts repository (Part:BBa_K1033922).
Traditional textile dyes often require heavy metal mordants in order to be efficiently fixed onto their cloth substrates (3). To avoid the use of these toxic mordants, we set out to fuse a cellulose binding domain (CBD) to the previously mentioned chromoprotein. CBDs are found naturally in β-1,4-exoglucanases which function to break down cellulose into cellobiose which can then be converted to glucose by β-glucosidases (4). The binding domain of the model CBD from Clostridium cellulovorans works by binding non-crystalline cellulose in a cleft on the surface of the protein which involves two tryptophan residues and several aspartate and asparagine amino acids (Figure 1). Furthermore, the dissociation constant of the same domain was experimentally determined to be 0.6 μM which suggests that the binding is extremely specific (1).
In our experiments, the cellulose binding domain from C. cellulovorans and Cellulomonas fimi were tested and parts for both were found on the iGEM parts repository from the 2012 Bielefeld-Germany, and the 2020 Waterloo teams respectively (Part:BBa_K863111 and Part:BBa_K863101).
To construct the plasmids of our fusion proteins, the CIDAR MoClo Kit was used from our team’s previous project. This was an obvious option to our team as we were already familiar with the 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.
Figure 1. Structural representation of the cellulose binding domain from Clostridium cellulovorans binding to noncrystalline cellulose (2).
We first adapted and combined the 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 proved to be much brighter than meffRed even under normal light. We also adapted an arabinose induced promoter to fit our cloning scheme to increase expression.
Our project after designing the gene fragments involved cloning these parts together and characterizing the resultant proteins on cotton fabric dyes. The rest of the parts listed on the engineering page are those expression vectors. We went through many unsuccessful combinations and iterations before finding a construct that produced a vibrant dye while still specifically binding to the cellulose in cotton. pGEC046 (BBa_K4258046) is the culmination of our work this year and will be the starting point of any further research.
References
- Goldstein, M. A., Takagi, M., Hashida, S., Shoseyov, O., Doi, R. H., & Segel, I. H. (1993). Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A. Journal of bacteriology, 175(18), 5762–5768. https://doi.org/10.1128
- Notenboom, V., Boraston, A. B., Chiu, P., Freelove, A. C., Kilburn, D. G., & Rose, D. R. (2001). Recognition of cello-oligosaccharides by a family 17 carbohydrate-binding module: an X-ray crystallographic, thermodynamic and mutagenic study. Journal of molecular biology, 314(4), 797–806. https://doi.org/10.1006/jmbi.2001.5153
- Patel, B. H. (2011). 11 - Natural dyes. Handbook of Textile and Industrial Dyeing (395–424). doi:10.1533/9780857093974.2.395
- Yeoman, C. J., Han, Y., Dodd, D., Schroeder, C. M., Mackie, R. I., & Cann, I. K. O. (2010). Chapter 1 - Thermostable Enzymes as Biocatalysts in the Biofuel Industry. Advances in Applied Microbiology (σσ. 1–55). doi:10.1016/S0065-2164(10)70001-0