Design

To enable the degradation of microplastics in a sustainable and eco-friendly manner, we have engineered bacteria to produce enzymes capable of breaking down PET molecules, PETase. With the additional introduction of Rhodococcus into our system, the products will be further degraded into H₂O and CO₂.

Introduction of PETase

IsPETase and BhrPETase 

Initially, during our experiments, we utilized E. coli as chassis. Its advantages of a short growth period and ease of genetic manipulation makes plasmid construction quicker and easier. However, E. coli is unable to generate energy independently. This makes cyanobacteria, Synechococcus elongatus PCC 7942, more suitable chassis for our purposes. Cyanobacteria are capable of harnessing solar energy through photosynthesis, reducing reliance on external carbon sources. This characteristic contributes to a more environmentally friendly and cost-effective PET degrading system. Additionally, regarding the impression of the final output, the appealing green color and the odorless nature of cyanobacteria, further establishing it as a more preferable chassis compared to E. coli.

The overall degradation mechanism is as follows.

The modified organisms containing genes that encode either IsPETase or BhrPETase synthesize these enzymes intracellularly. The addition of a signal peptide on the PETase assists the enzyme to exit the E. coli. Once outside the cell membrane, the PETase will degrade PET into monomers by breaking the ester bonds. The resulting products MHET, TPA and EG, will be further decomposed by the bacterium Rhodococcus. These bacteria will break down TPA and EG into CO₂ and H₂O.

Rhodococcus is bacteria widely used in environmental pollution bioremediation [4] , such as degradation of xenobiotic substances, heavy metal adsorption, reduction and bio desulfurization. Therefore, Rhodococcus is considered as one of the best candidates strain for in situ bioremediation. The Rhodococcus jostii is in charge of degrading TPA and EG, which were the monomers produced by the PETase degrading process, into H₂O and CO₂.  

To allow E. coli to produce PETase, we introduced the genes encoding these enzymes using plasmid vectors. We constructed our initial plasmids that were introduced into E. coli using pET28a, a classical plasmid vector used for protein expression in E.coli. This vector contains the T7 promoter, the lac operator, a ribosome binding site, the 6xHis sequence, and the T7 terminator. The T7 promoter is a strong promoter recognizable by T7 RNA polymerase, used to regulate gene expression of recombinant proteins. The lac operator can be activated by IPTG and used to control gene expression. The 6xHis sequence encodes for a tag that facilitates protein purification; however, we did not take advantage of it since our design does not require the extraction of the enzymes. Aside from the features included in the plasmid backbone, the genes coding for the different enzymes are inserted between the ribosome binding site and 6xHis sequence. In addition to using the two PETase from the iGEM distribution kit: IsPETase and BhrPETase. We applied an AI model we trained to generate mutated versions that possibly encode enzymes with higher efficiencies.

Figure 2. Table of mutated versions of IsPETase/BhrPETase

Upon consulting ChatGPT, we learned that in order to secrete the enzymes synthesized within the bacteria cells, we needed to attach signal peptides to the enzymes. ChatGPT provided us with several signal peptide sequences. We then confirmed to use the pelB sequence after conducting further research. This sequence is inserted before the PETase sequence.

Figure 3. Diagram of plasmid for E. coli

Subsequent to deciding to use cyanobacteria as our chassis, we rebuilt our plasmids using a transfer vector instead. We inserted the sequence including the signal peptide, PETase and 6xHis sequences between the promoter PpsbA2 and Bb_B0015. The t-vector transports the genes into the cyanobacteria, which are then incorporated into the host cell genome.

Figure 4. Diagram of plasmid for cyanobacteria