The aim of the dry lab team of iGEM IISER Thiruvananthapuram is to understand the nature of the chimeric protein engineered by the team. After an extensive literature survey that inspired our work, we came up with recombinant proteins capable of lysing the cell wall of fungi.
As the proteins are synthetically designed, we needed to understand their efficiency to bind to the chitin polymer of the fungi cell walls. Obtaining the predicted protein structure and docking the chimeric chitinases with chitin octamer gave us promising results in terms of binding affinity. The binding affinity values further provided us with the dissociation constant of the proteins, which was modeled to obtain the chitinase-chitin concentration at any given time.
Different species of fungi differ in their nature of growth. Modeling their growth to analyze growth rate would help us to determine how their growth has been affected while administering the protein.
Quantifying the activity of the protein at different physical conditions can help in characterising it. That is why we planned to work on various enzymatic models. Moreover, as mentioned above, different fungi grow differently, one would need to know how much of the protein would be needed to have an impact on their growth. So, we sought out models for IC\(_{50}\) and worked on a proxy molecule to validate the chosen models.
Given the time and effort needed to clone a gene of interest and purify the protein successfully, we need to know the optimum IPTG concentration needed to help in the induction of the protein. This would reduce the time of trial to get the right concentration of IPTG. Collaborating with Team iGEM Ecuador, we had successfully calculated the amount of IPTG that would be required for optimal protein production. Moreover, we also predicted the max amount of protein that a single E. coli cell would be able to produce and further went on to verify our predictions with experimental results.
As mentioned in our proposed implementation, we wish to use nanoparticles (PLGA) to release our chimeric chitinase into the human body. Administering the human body with a foreign particle, requires knowing the time for which the nanoparticle remains in the body system to anticipate any possible side effects. Thus we planned to model the degradation process of the nanoparticle under physiological conditions.
Thus, we have attempted to model every important step in characterizing our chimeric protein, providing promising results to further work on this project and data to help design experiments in wet lab.