Overview

Mathematical Modeling

The purpose of using PET TWINS is for the social implementation of PET degradation by BIND-PETase. To make this feasible, it is necessary to verify the biology in the wet lab, the safety system, and the potentials by the extension of the reaction system. In the dry lab, 7 model parts were created and each model is divided into 3 sections to provide the necessary validation and support for the implementation of PET TWINS.

In Section 1, we optimized the experimental conditions by analyzing the parameters that have a large impact on the desired behavior from the electrical-response module to PET degradation, which we have contributed to effective lab experiments. In Part 1, the electrical-response gene circuit was modeled, from the redox reaction of Pyocyanin and SoxR, Cre-Recombination, to the expression of BIND-PETase. Here, we succeeded in simulating the behavior of the electrical-responsive promoter, which had not been modeled by an iGEM team before, and proved that the promoter functions properly. Next, in Part 2, we modeled the extracellular transport of the expressed BIND-PETase and genome-derived csgA. Then, in Part 3, we formulated the formation of Curli Fiber in the extracellular space using the expression levels from Part 1 and 2. At last, in Part 4, the degradation of PET by BIND-PETase was evaluated. This represented a complex biological phenomenon using a simple mathematical and physical method that probabilistically models the cleavage of the PET macromolecule. This model is versatile, as it can be applied not only to BIND-PETase but also to other whole-cell catalysts.

In Section 2, since BIND-PETase is a whole-cell catalyst, we evaluated multiple sterilization methods, demonstrating the safety and advantages of using electro-sterilization. While in Part 1 we explained the expression of BIND-PETase, in Part 5, we added a secondary electrical response to express MazF, and evaluated the effectiveness by creating an artificial model of E.coli affected by MazF expression. From the results, we found that the use of this promoter requires careful control, which shares similar ideas where it is fragile to use. For Part 6, we compared the required energy for sterilization by means of electricity or heat, and found that electricity is far more efficient than heat.

In Section 3, we examined the effect by scaling up the reaction system for social application. In Part 7, we examined the conditions necessary for adequate diffusion of Pyocyanin, used in the electrical-responsive promoter in Part 1, on a larger scale. We succeeded in simulating diffusion using the CIP method in three dimensions, which is often done in two dimensions and for which there are few previous studies. This model enables us to easily simulate the expansion of a reaction system on an industrial scale. In addition, it can be applied to substances other than Pyocyanin by simply changing the constants, which will be a significant contribution to the iGEM community.

In Silico Simulation

In the Simulation Part, we firstly constructed an In Silico Evolution Pipeline to evolve PETase. The In Silico Evolution Pipeline is thoroughly documented, and the code is publicly available, making it highly versatile—users only need to change the target protein to evolve. This setup contributes significantly to the iGEM community, as it allows other teams to easily replicate the process.

In our project, we designed multiple proteins not only through the In Silico Evolution Pipeline but also through Rational Design. Next, we performed Protein Characterization in silico. We successfully evaluated the properties of PETase variants not only through wet-lab experiments but also from in silico simulations. Our new composite part, BBa_K5436124, was evaluated using in silico simulations to assess its structural stabilities. For more details, please refer to BBa_K5436124.