All organisms in nature produce pigments. However, those are usually organic molecules synthesized through complex pathways. Anthozoa are an exception, as several species belonging to this class produce proteins that are intrinsically colored1. These chromoproteins are homologous to GFP and they can either be fluorescent or non-fluorescent. Non-fluorescent proteins are colorful in visible light, as shown by the fact that coral colors are determined by chromoproteins instead of other organic pigments2.
As chromoproteins are coded by single genes, it is relatively easy to produce them with synthetic biology compared to engineering a whole pigment production pathway. Also, given their protein nature, they can be fused to other proteins. This has historically been done with GFP. However, in the past few years, the necessity of having more colors available has lead to an increasing characterization of other chromoproteins2.
The scientific interest in chromoproteins has been so far directed towards their use as reporters. Indeed, when a fluorescent protein gene is fused to a gene of interest, the detection of fluorescence is an indicator of the expression of that particular gene. In our project, we are interested in producing chromoproteins not as a reporter, but to make use of their color itself. By fusing them with a cellulose binding module, a keratin binding domain, and silk repeats in different constructs, we aim to use chromoproteins as dyes.
The chromoproteins we chose for this project are PrancerPurple, mRFP, and sgBP. We chose PrancerPurple based on the good results of its thermostability3. RFP was chosen because a monomeric version was available and we were interested to find out if the smaller monomeric RFP folds and functions better compared to the normal chromoproteins. The blue chromoprotein sgBP was chosen because it was not found as a BioBrick. Unfortunately, the sgBP sequence did not work as we anticipated, so we could not submit it as a BioBrick and dropped it from the further experiments.
1 Kelmanson, I.V. & Matz, M.V. (2003) Molecular Basis and Evolutionary Origins of Color Diversity in Great Star Coral Montastraea cavernosa (Scleractinia: Faviida). Mol. Biol. Evol. 20(7):1125–1133. doi: 10.1093/molbev/msg130
2 Alieva, N.O, Konzen, K.A., Field, S.F., Meleshkevitch, E.A., Hunt, M.E. et al. (2008) Diversity and Evolution of Coral Fluorescent Proteins. PLoS ONE 3(7): e2680. doi:10.1371/journal.pone.0002680
Spider silk consists of repetitive sequences which account for often more than 90% of its structure. Repetitive sequences are located in the middle of the whole spider silk protein. The terminals of the protein are nonrepetitive and assemble spider silk proteins into fibers. Spider silk has unique properties. It has high resilience and it is not only strong but also elastic1. However, spiders can't be farmed, which makes spider silk production difficult2. Spider silk has been produced synthetically in several host organisms, such as bacteria, yeast, plants, and silkworms3.
Binding with CBM and KBD
Natural dyes have problems with the color fading. To solve that, we created constructs with chromoproteins linked to binding domains. With the help of the binding domains, chromoproteins will bind to the desired surfaces better and stay on them longer.
The cellulose binding module we used is CBM3 from Clostridium thermocellum that is thermostable in high temperatures, which makes it ideal to use with our heat-dependent purification protocol. CBM enables the binding to cellulose-based materials such as cotton in our textile-oriented approach. Cellulose-based materials are used in many forms, e.g. plastic-like materials, which makes it possible to use our dyeing method in other applications than just textiles.
We wanted to expand our dyeing method to other materials than cotton. For this purpose, we found the keratin-binding domain KBD-C. KBD is one of the three keratin-binding domains from human desmoplakin which have been shown to bind to epithelial cytokeratins and vimentin1. The structures of cytokeratin and for example hair keratin are similar, which is why KBD of desmoplakin is expected to bind also to hair2.
In our experiments, we tested how the chromoprotein with KBD would bind to human hair, which would further expand the applications for which we could use our dyeing method.