Enzymatic Data

Looking to the future, ReneWool plans to thoroughly test the enzymatic activity of our cellulase and keratinase and compare the results to those predicted by our software. With this goal in mind, we would complete engineering of the cellulase Cel-CD by cloning it under a constitutive promoter and transforming it into Escherichia coli. We aim to calculate the Michaelis-Menten parameters of Cel-CD and KerDZ and identify the binding interactions of the enzymes with pure cellulose and keratin first, and later with cloth of varying fiber composition. To accurately measure enzymatic degradation of waste products, we plan to implement the reporter system we designed which involves mScarlet—a fluorescent domain—krt31 acting as a linker, and RShadow, a domain that blocks the fluorescence from mScarlet. This reporter system involves a quenching system whereby the activity of KerDZ in solution is inversely correlated with the intensity of fluorescence. When KerDZ degrades the krt31 linker, ShadowR is detached from mScarlet. This system was inspired by the 2023 UAlberta iGEM team's Q-sensor iGEM Parts Registry
. This GEM would provide an easy, quick, and cost-effective method of determining enzymatic activity qualitatively. However, for more accurate and direct quantification of keratin, we may use assays, MicroScale thermophoresis, or co-immunoprecipitation, however these methods will have a cost barrier. For cellulose degradation, simple sugar assays will likely suffice.

After in-depth data collection and analysis, we plan to insert KerDZ and Cel-CD into a singular plasmid under a constitutive promoter. Following successful cloning and transformation into E. coli, we plan to use similar methods to compare protein expression and secretion to KerDZ and Cel-CD expressed individually. To create this large plasmid, further research and development will be done to identify the optimal constitutive promoter, Ribosome Binding Site (RBS), and a suitable terminator. Fortunately, our previous efforts in designing, building, and testing various Upstream Regulatory Sites in different vectors gives us a foundation of learning to work with.

Expression System

Of great significance to the proposed system is the ability to induce the expression of Spider Silk using the product of cellulose or keratin degradation. For this reason, Spider Silk will be cloned under an inducible promoter which can be activated by the degradation product of cellulase or keratin, linking their degradation to the production of Silk. This will likely be linked to cellulose degradation, as the products, disaccharides/polysaccharides, are commonly used as inducers. This ambitious system will require thorough investigation. Finally, we plan to transform this plasmid, containing spider silk, into a culture of E. coli that contains the previous plasmid containing KerDZ and Cel-CD. If successful, we will have created a single bacterial culture capable of secreting enzymes to degrade waste products and control the production of Spider Silk. Contingencies have been considered if this proves too cumbersome for E. coli, such as experiment with different microorganisms, such as Saccharomyces cerevisiae for its more complex machinery, supplementing the culture with helper tRNA for glycine and alanine (common amino acids in our Spider Silk protein), or even integrating the genes for degradation into the organism's genome to reduce the burden of multiple recombinant plasmids.

Increasing Scale

Next steps should involve exploring the upscaling process to take the system from shaker flasks to bioreactors. Considerations should be taken to capture greenhouse gasses (such as CO₂) and reduce emissions. Interest has been taken in brewing companies that implement AI and new technology to capture the pure CO₂ emissions, reducing Greenhouse Gas emissions, and earning additional revenue.¹ To accomplish scaling-up and optimization we will monitor the inputs and outputs of the system and fine-tune each aspect of biomass accumulation and protein production. Additionally, several bioreactor designs must be built and thoroughly tested including, but not limited to, media conditions, O₂ rate, temperature, pH, etc. and a variety of different keratin and cellulose containing textile wastes to examine the degree of pre-processing required for different common waste types. We plan to test the efficacy of precipitating Spider Silk after production as a cost-effective method of extracting our product. Alternatives involve membranes calibrated to the physical properties of the desired protein such as size, charge, or polarity to develop an effective purification system.

Future Considerations

Past literature review and expert interviews have pointed out the inefficiency of Spider Silk production in E. coli and if our results are poor, we may begin looking for alternative valuable recombinant proteins. An interesting avenue that merits further research is the production of Hagfish intermediate filaments, composed of two proteins, denoted as α and γ, which are comparable in mechanical properties to natural dragline silk from orb‐weaving spiders, and has been produced in significantly greater quantities than the well-studied spider silk.² As this valuable and strong material contains significantly less repetition than the gene for spider silk, it is likely that E. coli is able to produce Hagfish intermediate filaments at a significantly greater rate, allowing for sooner return on investment (ROI) and a greater supply to be used in various medical and commercial industries.

Finally, our vision for ReneWool involves taking it a step even further. We plan to one day create a modular and universal system that can be customized to produce different proteins as desired from various streams of textile waste.