Part:BBa_K5236008

IsPETase-S121E/R224Q/N233K

Because of the substantial amount of microplastic pollution contaminating our oceans, our project aims to use biosynthetic methods to achieve effective degradation of these compounds in ambient temperatures. Whilst there are existent chemical and biological measures for microplastic degradation, however, chemical means often release chemical byproducts and contaminate the environment, whilst enzymatic degradation can only be done in an environment with specific pH, temperature, and humidity levels.[1] IsPETase-S121E/R224Q/N233K comes from Lu. et al. 2022 but is constructed by us in Escherichia coli. It breaks down polyethylene terephthalate into terephthalic acid through hydrolysis. IsPETase is an enzyme found in Iodeonella sakaiensis that possesses the ability to degrade PET[2].

Fig.1 The IsPETase-S121E/R224Q/N233K protein structure predicted using Alphafold and ligand binding predicted using Autodock, mutation sites are marked in red in the images.

Usage and Biology

IsPETase is an esterase, specifically designed to break down ester bonds between polyethylene terephthalate, a polymer, into terephthalic acid, a monomer. IsPETase-S121E/R224Q/N233K is a mutated sequence of PETase which enhances its degradation efficiency. It contains mutations at sites S121E, R224Q, and N233K. The mutation at position 121 involves a negative side chain, which enhances its catalytic efficiency. The mutation at position 224 replaces arginine with glutamine, facilitating better substrate access. The mutation at position 233 replaces asparagine by lysin which increases the thermal stability. The mutations were derived from a Transformer model on 1007 homologous PETase protein sequences derived from the UniProt Database using the masked language model (MLM) training method. For further information, please check the  model page on our wiki. https://2024.igem.wiki/basis-china/model

The IsPETase-S121E/R224Q/N233K sequence is expressed in E.coli using the pET28a vector. The pET-28a is 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. Asides from the features included in the plasmid backbone, we added a signal peptide sequence — pELB — before the BhrPETase N205G sequence, which is inserted between the promoter and terminator.

Our experimental design was aimed at proving the functionality of our mutated sequences, the viability of E.coli while harboring our sequences, the translational capacity of E.coli and the successful degradation of our mutated enzyme. To validate the functionality of our mutated sequences, we first inserted our genes into plasmid vectors. This process was initiated with the design of specific primers, followed by PCRs. Then, our sequences were recombined into plasmids and transformed into the chassis. In order to assess the part sequences for the mutated IsPETase variants S121E/R224Q/N233K and the functionality of the enzyme, we designed our experiment into two sub-parts. The first is plasmid construction. The second is to test the enzymatic activity. Through conducting colony PCR, we tested whether there has been successful transformation of our parts into the chassis. The following result of electrophoresis proved this since the sequence containing our mutated genes has a total of 891 base pairs with the sequence at the corresponding locations of the marker.

Fig.2 The DNA gel electrophoresis result.

After confirming the integration of IsPETase into our chassis, we proceeded to test whether E.coli could maintain its viability while harboring our genes. Thus, we cultured the bacteria on a petri dish. After a 24-hour incubation period, E. coli grew over the plate, indicating that it can harbor our genes.

Fig.3 The result of plate coating. IsPETase-S121E/R224Q/N233K was coated on spectinomycin plates and incubated overnight at 37 ℃.

We assessed the translational capacity of E.coli and the efficiency of our mutated enzyme. This section presents an analysis of two key results. 1. SDS-PAGE Electrophoresis: the electrophoresis result of our protein validates that our enzyme can successfully synthesize the proteins. 2. Enzyme Kinetic: the dynamic curve of our enzyme shows a high efficiency in degrading rate, in which S121E/R224Q/N233K, M10L, H104W, W159H/F229Y outperformed WT IsPETase. The relative enzyme efficiency (A415/protein concentration) that we are looking at takes into consideration both the efficiency of the enzyme itself and the PETase synthesis rate of the chassis, since our end goal is to implement the engineered organism in a self-sufficient PET degrading system as a whole.

Fig.4 SDS-PAGE electrophoresis result.The Cell Culture group demonstrated the expression of the proteins, while we also tested the medium where cells were centrifuged and discarded. Proving the presence of IsPETase extracellular.
Fig.5 The enzymatic activity of mutated IsPETase was measured using p-nitrophenyl butyrate. The results showed that the IsPETase-S121E/R224Q/N233K have approximately 2.5-fold higher activity than WT group

After confirming the enhanced efficiency of our enzymes, we have reached the most essential part of our part examination, which is to test if our mutated enzyme is capable of degrading microplastics. We designed two main approaches to validate it. First, we employed the scanning electron microscope to observe the morphological changes of the plastic after exposure to E.coli with IsPETase. The results confirm the degradation capacity of the enzyme, as there is a notable change on the surface of the plastic. However, observations are insufficient to prove the effectiveness of our enzymes. Consequently, we conducted an additional experiment utilizing High-Performance Liquid Chromatography (HPLC). We measured the amount of TPA in the cell culture medium, the peak of IsPETase S121E/R224Q/N233K in E.coli and cyanobacteria reflects the peak of the TPA standard sample, which proves the effectiveness of the microplastic degradation capabilities of our enzymes. To conclude, IsPETase-S121E/R224Q/N233K (891 bp) has been successfully transformed into E.coli.

Fig.6 S121ER224QN233K Electron Scanning Microscope result
Fig.7 The HPLC results after 14 days of incubation of IsPETase-S121E/R224Q/N233K and PET. The results showed that the concentration of TPA, a degradation product of PET, was elevated in the cultures compared to the control group, implying that the degradation of PET

After determining that our enzyme IsPETase-S121E/R224Q/N233K expressed in E.coli is actually more efficient than the wild type IsPETase, we moved on to expressing the gene in Synechococcus elongates PCC 7942. To achieve this we inserted the signal peptide and IsPETase-S121E/R224Q/N233K sequence between the PpsbA2 and terminator Bba_B0015 from a transfer vector.

Fig.8 The result of IsPETase-S121E/R224Q/N233K DNA sequencing, the results showed that IsPETase mutation and signaling peptide pelB sequences have been correctly assembled.

The reconstructed plasmids were transformed into cyanobacteria and coated on BG11 plates containing the antibiotic spectacularionomycin. Single colonies containing the IsPETase-S121E/R224Q/N233K gene appeared after two weeks of incubation.

Fig.9 IsPETase-S121E/R224Q/N233K in cyanobacteria coating result

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

Lukaszewicz, Paulina, et al. "Plastic Waste Degradation in Landfill Conditions: The Problem with Microplastics, and Their Direct and Indirect Environmental Effects." International Journal of Environmental Research and Public Health, vol. 19, no. 12, 2022, pp. 12345-12367. National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9602440/.

Lu, Hongyuan, et al. “Machine Learning-Aided Engineering of Hydrolases for Pet Depolymerization.” Nature News, Nature Publishing Group, 27 Apr. 2022, www.nature.com/articles/s41586-022-04599-z.