Successful Cloning and Color Production
The first stepstone of our project was to successfully clone together a set of disparate parts to construct a functional expression vector. While some of the parts we used were already established plasmid parts in the MoClo cloning scheme, all of our coding sequences and our arabinose inducible promotion system were adapted from existing iGEM parts to be compatible with the MoClo cloning scheme and the design of our project. Our first attempts at golden gate cloning were unsuccessful. Our transformation plates had very low efficiency indicating the plated cells did not have the kanamycin resistance that our Golden Gate construct would confer. Additionally the transformation plates had no pink colonies.
After ordering modified versions of our (1)-(5) CD sequences, we tried the Golden Gate cloning again and began to see color in both our meffRed and mScarlet colonies. Seeing the color produced in our colonies not only indicated that our Golden Gate reaction was successful, but that we produced a protein that correctly folded to express color as well. The colonies that should have produced the meffRed chromoprotein only produced a very faint pink color, which was contrary to the results of the 2013 Uppsala iGEM team that we adapted the sequence from. They showed a more vibrant and darker-hued red color. This could mean there was an error in the sequence adaption. Fortunately, the mScarlet sequences that we designed produced extremely vibrant pink colonies, and we decided to move forward with the well-performing mScarlet.
Interestingly, pGEC046 with the CBDfimi domain produced a much more vibrant pink pellet than pGEC049 with CBDclos. The difference in vibrance could be explained by any differences in the energy cost of protein biosynthesis between CBDclos and CBDcex. pGEC046 and pGEC049 also have different orientations which could impact protein folding via different proximity of the sequences’ components and the location of the carboxyl and amino terminuses.
Cotton Binding Affinity
Once we solidified our ability to produce the mScarlet protein, we moved on to testing the differences in our synthesized CD parts((16)-(20)). The different CD sequences in our pGEC046-pGEC050 constructs were meant to allow our team to evaluate differences in cellulose binding domains and orientations. Unfortunately, our (18) CD sequence was not designed correctly so we could not compare all of the combinations of binding domains and orientations as we had hoped. Instead, we primarily compared our brightest strain with a cellulose binding domain (pGEC046) to our mScarlet negative binding control (pGEC050). pGEC046 and pGEC050 are identical constructs except that pGEC050 lacks the CBDfimi cellulose binding domain present in pGEC046. After lysing the cells and removing the insoluble material, it was realized that most of our protein was in inclusion bodies - significantly reducing protein yield. Due to a lack of time, we could not optimize a protocol for re-solubilizing and re-folding. The lysate was still standardized and used to dye pieces of a cotton t-shirt. This was a qualitative method that was based on visual changes in brightness. Looking at the plain images there is only a slight difference. However when the brightness is lowered, there appears to be a more significant difference. These results are qualitative and preliminary, but promising.
Experimental results of the wash experiment. Pre-wash (top left), post-wash (top right), pre-wash with reduced brightness (bottom left), and post-wash with reduced brightness (bottom right).
Application
The goal of our project was to work towards creating a sustainable protein-based alternative to azo dyes. We have made substantial progress in this objective. The first step made towards a dye is the production of recombinant proteins. MeffRed was not an effective chromophore when combined with the cellulose binding domain. This was replaced with mScarlet, and a successful protein that contained the chromophore and binding domain was produced. Not only was it produced, but it was produced in a high quantity. Our design of combining a binding domain with a protein chromophore is also a concept that could be implemented and used for other materials. For example, using keratin binding domains for hair dyes. Our project represents the creation of a modularly created recombinant protein dye relying on a chromophore combined with a binding domain. We have proved this system can work, opening the door for many other combinations of chromophore and binding domain.
The binding affinity for this recombinant protein has yet to be quantitatively verified, but the preliminary data is promising. This is especially true since we were working with impure lysate that would only decrease binding affinity due to charged compounds and other potentially destabilizing factors present. There are multiple easy steps that could be taken to obtain pure lysate due to the inclusion of the 6x-His tag. Overall, there are many steps left before we create a usable product, but substantial steps have been taken toward that goal.
Up Next
As mentioned earlier there are many steps necessary before making our construct into a product. Here a list of potential steps will be outlined that would represent the path from our current prototype to a functional product. Some immediate steps to be taken include 6x-His tag purification and protein concentration. Doing these steps would allow pure protein to be used to determine the dissociation constant, as well as other physical properties of the recombinant protein. Similarly, it will be important to pursue physical characterization and determining properties such as thermostability, an important trait in the clothing dye industry. Once the dye is purified and fully characterized, it can be improved. This would involve developing a re-solubilization and re-folding protocol, or developing a culturing method that doesn’t produce inclusion bodies. Next, improving yield would rely on optimization of protocol or potentially using error-prone PCR to create a library of genetic variants, then screening the library to identify superior mutations. Once all of these steps are completed, the end product would be a plasmid that efficiently produces a cellulose binding dye with improved physical properties.