Literature
In this iGEM event, we confirmed through literature research that Rhodococcus opacus PD630 can use aromatic compound accumulation to produce microbial lipids. Additionally, we found that PETase and MHETase can degrade PET plastic. Throughout our experiment, we made progress in this area. We attempted to utilize Cupriavidus necator H16 to convert the TPA resulting from degradation into PHB, a biodegradable plastic that can serve as a sustainable alternative to PET plastic. However, our team needed to conduct comprehensive research in this area. Therefore, iGEM could explore and pursue this avenue in the future.
The literature review found that PETase can cleave ester bonds in PET, leading to the hydrolysis of polymerized PET molecules, which generates both the dimer BHET and the monomer MHET. PETase contains an α-β-hydrolase fold with a twisted nine-stranded β-fold flanked by six α-helices. Moreover, the catalytic triad in PETase includes Ser-His-Asp residues. To further elaborate, PET molecules are hydrolyzed via a nucleophilic attack on the Ser residues of the PETase catalytic triad. The molecule's hydrolysis comprises a nucleophilic attack by the Ser residues of the PETase catalytic triad.
MHETase cleaves the ester bond in MHET, breaking it down into terephthalic acid TPA and ethylene glycol EG. The catalytic triad of MHETase consists of Ser-His-Asp residues, which catalyze MHET via a ping-pong mechanism-based acylation-deacylation process. The active site of MHETase has an α-β hydrolase fold. Technical terms have been explained, and complex terminology has been avoided for clear and concise communication. Passive voice and impersonal construction have been used for objectivity while ensuring grammatical correctness and precision in word choice. The Ser residue initiates a nucleophilic attack on the MHET molecule, leading to the formation of the enzyme-acyl intermediate and the removal of the glycol fraction. Deacetylation is activated by water molecules that enter the active site following the removal of the glycol, resulting in the production of the end product, TPA.
So, this literature review forms a solid theoretical foundation for the subsequent alteration of PETase via targeted mutation and model construction.
Additionally, our team has introduced a Tph manipulator in this project, which converts TPA into PCA. PCA can swiftly enter the tricarboxylic acid metabolism accumulation and produce microbial lipids through the β-ketoadipic acid pathway. This theoretical design can be expanded to modify other chassis organisms.
Basic elements
In this year's iGEM project, our team created a sequence of pBSKR7756 vectors suitable for erythrocystis expression. We ligated PETase, a PETase-MHETase fusion protein, mCherry fluorescent protein, and Tph operon and identified the optimal signal peptide, enhancer, and promoter. Future iGEM teams can utilize these developments. We conducted experiments to investigate the ideal Rhodococcus opacus PD630 induction conditions. These manipulations can be used as guidance by future iGEM teams when selecting Rhodococcus opacus PD630 as a chassis organism.
Wiki and Video
As WUST-China was the first team to participate in iGEM, we have successfully created our initial wiki this year. Building the wiki and its webpage has provided a model and guidance for succeeding iGEM teams. The WUST-iGEM teams can progress the wiki we have created this year through revitalizing and innovating. We have also produced promotion and presentation videos for the first time. Similarly, the WUST-China team has made promotional and presentation videos for the first time. We can offer comprehensive guidance and experience to future WUST-China teams throughout the entire video production process.
Human Practice
The WUST-China-2023 team faced challenges during its first competition, particularly in HP activities. From the initial handicap to subsequent methodical approaches, our team has been continuously learning. Through exchanges with other iGEM teams, we have gained knowledge and experience in HP activities, which has helped us to actively promote inclusiveness, breadth, and diversity in our approach. We started to actively stay in contact with other iGEM teams, which offered a platform and connection for succeeding iGEM teams and iGEM teams from different schools; moreover, achieving iGEM teams was more specific and comprehensive in their HP interests. Through our experiences this year, we have offered guidance for future WUST-iGEM teams to conduct cooperative efforts and exchanges and to promote and publicize science within and outside the school.
Structure of team members
Our initial member structure may have needed to be more rational and methodical as a novice team. During the competition, we adjusted members' roles and responsibilities. Participating in this year's iGEM tournament has dramatically enhanced our comprehension of the member structure. This newfound knowledge will be a beneficial reference for recruiting future iGEM team members.
At the same time, we also develop through practice. Each iGEM team member should have involvement in every aspect of the experiment operation. It aids in promptly understanding the project's principle and also contributes towards accelerating the progress of the experiments.
iGEM Participation Experience
In this year's initial iGEM tournament, we have acquired practical experience and comprehension of every aspect and time point. We have gained in-depth knowledge of tournament specifics, including wiki building, video production, poster creation, and IGEM component uploading. This invaluable first-hand involvement will be introduced in the December 2023 training for the new iGEM squad, enabling future iGEMs to continue building on this year's tournament.
