iGEM Stockholm 2018 Wiki

Demonstrate

After spending time investigating what problems can be solved through synthetic biology, we landed on the track of helping wastewater treatment facilities solve the issue of degrading pharmaceutical residues before they reach the environment. As a solution, we developed an innovative enzymatic approach towards tackling this problem. We built a method incorporating a model that can evolve from experimental analysis to improve the specificity of a fungal laccase towards a specific antibiotic. As a proof of concept, we have demonstrated our work with sulfamethoxazole – one of the persistent antibiotics found in the Baltic Sea region. We then immobilized the laccase on magnetic beads and progressed out of the lab. We designed a prototype to demonstrate how our product can be implemented in wastewater treatment plants; whereby the biggest contributing factor to the design was our many visits to wastewater treatment facilities and companies within the field to investigate and improve on our implementation.

Figure 1. A summary of the methodology and implementation of Biotic Blue.

1. Can Laccases Reduce the Toxicity of Sulfamethoxazole (SMX)?

Before we go further with our concept, it was important to prove that laccases can degrade SMX to products that are not ecotoxic and hence safe for the environment. Through ecotoxicity testing with a commercial laccase and the wild-type laccase that we produced, we were able to conclude that SMX ecotoxicity was reduced and that the reaction products are safe.

2. Building Our Production Platform

After thoughtfully choosing our production organism, we successfully produced the wild-type laccase from Trametes versicolor in Pichia pastoris.

3. Building Our Platform Technology

We have successfully immobilized the laccase on magnetic beads. The following images show that the immobilized enzymes were functional (Figure 2.1) and that the magnetic beads can be captured and separated from water solution via a magnet (Figure 2.2).

Figure 2.1. On the left: Control. On the right: Immobilized laccases showing activity (color change) when ABTS (a laccase model-substrate) was added.

Figure 2.2. Recovery of immobilized magnetic beads on a magnetic rack.

4. Enzyme Engineering: An Iterative Evolutionary Approach

Figure 3. Summary of the Design, Build, Test Cycle as the engineering methodology used to integrate between modeling and experimentation.

As elaborated on our Project Design page, the main objective of engineering the wild-type laccase was to increase its specificity towards SMX to improve its degradation rate. Multiple engineering strategies can be performed, and we have explained why we specifically chose rational enzyme design for this project. Ultimately, this went through 3 phases:

Phase 1. Modeling Reaction Mechanism

The first phase of rational enzyme design was to find the reaction mechanism for SMX with laccases and the parameters that can describe it. As part of publishable work, we were able to model the reaction mechanism of our enzyme.

Phase 2. Validating the Model

The second phase was to understand and learn about our system starting with creating a valid model for the wild-type laccase. Through our simulations, we were able to determine how the laccase behaved in solution and how it interacted with a given substrate. We then inferred under which conditions the system operates and after that designed experiments that can directly prove or disprove our claims from the model. This was the first cycle of our design, build, test methodology (Figure 3):

Design: From the model we were able to calculate theoretical values of the catalytic parameter k_cat.
Build: We were successful in expressing, producing, and purifying the wild-type laccase from P. pastoris.
Test: An activity assay with the model-substrate ABTS was conducted to calculate experimental k_cat.

As a result, we succeeded in validating our model as the experimental k_cat value confirmed the theoretical value of k_cat for the model-substrate ABTS. This also standardized our pipeline with assays development for analysis, where mutants can be similarly tested for validation and then compared with the wild-type laccase (more can be found on our Integration & Analysis section).

Phase 3. Creating a Mutant

This phase describes the evolutionary iteration of our design, build, test methodology towards creating successful mutants.

From our first simulation of the mutated enzyme the design focused on increasing the catalytic activity of degrading SMX through making it fit better into the active site. We then successfully transformed the selected mutant into P. pastoris. However, in the testing stage (Figure 3) the enzyme was inactive and we inferred it could be due to misfolding. This, in turn, caused us to iterate back into the design stage (Figure 3) to test this assumption. As a result, it gave us a better insight on the folding modes of our simulated protein as our assumption was confirmed. Thus, this helped us to achieve our objective by expanding our model and introducing another generation of mutations, where the modeled k_cat was 10 times better than the wild-type k_cat. In the future, the cycle can be continued to express and test the next generation of mutants (Figure 3).

5. Product Design & Implementation

For the engineered laccases to be introduced at wastewater treatment plants (WWTPs) there needs to be a way for them to be captured and reused. That is why we developed a strategy of immobilizing laccases to magnetic beads so that they can be recovered by leveraging magnetism. After utilizing advice from experts, the following figure explains how our solution - Biotic Blue, can be implemented in WWTPs: