Results

Absract

Contribution

Parts

Enginering Success

Absract

In our ever-evolving world, where plastic pollution poses a formidable challenge, our team stands undeterred, united by a shared thirst for knowledge and a commitment to making a difference. As we participate in the IGEM competition for the second time, our focus remains sharp: addressing the pervasive issue of polyethylene terephthalate (PET) pollution. PET, a widespread polymer foundational to countless consumer products, particularly in packaging, lingers in our environment, refusing to degrade for centuries.

Despite the daunting challenges and our previous bouts with the limitations of knowledge, our passion has served as our North Star, guiding us forward. Our collective determination has brought to our attention the incredible potential of two enzymes: PET hydrolase (PETase) and mono(2-hydroxyethyl) terephthalate hydrolase (MHETase). Working synergistically, these enzymes can decompose PET into its basic building blocks, offering a revolutionary solution to the persistent issue of plastic pollution.

Our journey is centered on enhancing these enzymes' capabilities. By understanding existing mutations and exploring the potential for new ones, we aim to create a biocatalytic process that is both efficient and transformative. We picture a world where waste PET is not just discarded but is seen as a valuable resource, thanks to a process that can convert this waste into indispensable raw materials.

Contribution

In our mission to address PET degradation, we understand the value of building upon established knowledge. Rather than starting afresh, we're diving deep into previously discovered mutations, harnessing their insights to advance our work. That is why our team started from modelling and analysing the efficiency of mutations for degradation of plastic materials. We successfully performed a comparative analysis of various mutations including S238F, R61A, L88F, R280A, I179F, S121E, D280A for PETase enzyme and Phe424D, Phe424H, Phe424V, Phe424E, R411K for MHETase enzyme.

Our analysis showcased that, at 37°C with concentrations of 10 μg/mL, the combined action of PETase and MHETase resulted in a 40% reduction of a 100 μm PET film in 24 hours. Crucially, mutations such as S131G, E226T, and F495I impacted degradation negatively, underscoring their pivotal role in enzymatic action. This combined enzymatic approach, notably more effective than PETase alone, hints at the vast potential for efficient PET recycling, emphasising the importance of these specific mutations in the degradation process.

Parts

In biotechnology research and innovation, a key focus should be the potential modifications of the enzymes PETase and MHETase, which play an important role in the degradation of polyethylene terephthalate (PET). To this end, our team has developed a significant number of parts and constructs this year, although not all have been fully validated due to time and resource constraints

Our main goal was to clone the studied genes into appropriate vectors for expression in Pseudomonas putida. There are several plasmids with dual expression systems that can be compatible with Pseudomonas putida for co-expression of multiple genes. So, 2 vectors J435500 and pSEVA-25 were used to clone PETase and MHETase genes. During the research process, we have created and provided many designs and parts that, although not fully tested, may be useful to future researchers. We hope that our work will be a starting point for further research and bring new ideas and approaches to the study of plastic decomposition.

Figure 1: Map J435500 vector with differentially cloned genes.

Figure 2. Map of pSEVA-251 dual expression plasmid with CJ-MHETase and PETase genes.

Engineering Success

We had to overcome significant challenges on the way to beginning our experimental work. In the beginning, we were faced with waiting for permission from the university, which took more than four months. This wait was not an easy ordeal, requiring patience and perseverance, but we moved forward steadily, eager to begin our work. At the same time we had limited resources and were not able to order MHETase enzyme to fully test out our constructs. So, we tried to use everything available to conduct experiments and came up with the idea to build a part out of materials we had in the Distribution Kit 2023.

So, we used BBa_K2910000 and BBa_J435500 parts to test out the protein expression and enzymatic activity of IsPETase-W159H-S238F with C-terminal Hexahistidine Tag and created a new part BBa_K4901036 (http://parts.igem.org/Part:BBa_K4901036).

Emphasis was placed on studying the activity of the BBa_J435500_W159H_S238F variant compared to the wild-type enzyme. The experiment focused on the hydrolysis of pNPB at a substrate concentration of 2500 µM, at a slightly alkaline pH of 9.0 and a temperature of 30°C. The results of this part of the study showed a significant difference in enzymatic activity between the two variants.

The results demonstrated a remarkable difference in enzymatic activity between the two variants. Wildtype PETase: The wildtype PETase exhibited a relatively lower enzymatic activity under the specified conditions. The graph representing the wildtype PETase's activity showed a gradual increase in product formation (p-nitrophenol) over time. However, the rate of reaction was comparatively slower. BBa_J435500_W159H_S238F: In contrast, the variant BBa_J435500_W159H_S238F displayed significantly enhanced enzymatic activity. The graph depicting its activity revealed a much steeper slope, indicating a rapid conversion of pNPB into p-nitrophenol. The variant exhibited a catalytic efficiency that was seven times higher than that of the wildtype PETase under the same conditions. In our research, we meticulously engineered dual expression plasmids harbouring both PETase and MHETase enzymes. The objective was to optimise PET degradation efficacy, with an eye on scalability for potential implementation in large-scale PET degradation facilities. This endeavour aimed to facilitate enhanced PET recycling by leveraging the synergistic capabilities of these enzymes. Particularly notable was the BBa_J435500_W159H_S238F variant of PETase, which showcased a sevenfold efficiency increase compared to its wildtype counterpart. Despite challenges, we innovated with available resources, setting the stage for scalable PET degradation solutions in industrial settings. Our findings illuminate a promising avenue for sustainable plastic waste management in the future.