Part:BBa_K5236008

IsPETase-S121E/R224Q/N233K

This basic part encodes mutated IsPETase-S121E/R224Q/N233K, which comes from academic essays but is constructed by us in Escherichia coli. IsPETase is an enzyme found in Iodeonella sakaiensis that possesses the ability to degrade PET[1].

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

To insert our parts into plasmids, we’ve designed primers and performed PCRs. Then, our genes were recombined into plasmids and transformed into chassis. To test if our part codes for the mutated IsPETase S121ER224QN233K we want and whether the enzyme works, we've completed two large experimental processes. The first step is plasmid construction. And the second is to test the enzymatic activity.

By conducting colony PCR, we are able to test if our parts have been transformed into chassis successfully. The following result of electrophoresis proves that we’ve inserted genes into chassis since the sequence containing our mutated genes has a total of 891 base pairs and the results are in the right location.

Fig.1 The DNA gel electrophoresis result
Fig.2 The result of IsPETase S121ER224QN233K DNA sequencing

After proving that our genes existed in chassis, we need to test if the bacteria can survive as usual with our genes. Thus, we’ve coated the bacteria on nutritional petri dish. And after a night, E. coli grew over the plate our plate, justifying that E. coli can survive with the gene of our part.

The result show that chassis carrying our PETase could survive.

Fig.3 The result of plate coating

We tested whether the bacteria could translate for our protein, and we examined whether our mutated enzyme is more efficient. For this section, we analyzed two results as well. First, the dynamic curve of our enzyme shows its high efficiency in degrading rate. Second, the electrophoresis result of our protein proves that our enzyme can be successfully coded by the parts we designed.

Fig.4 Mutated IsPETase Dynamic Curve
Fig.5 Protein electrophoresis result

After proving that our enzyme are more effiicient, we moved on to test the ultimate and the most essential part of our part examination, which is to test if our mutated enzyme can actually degrade plastics. For this large step of process, we also designed two approaches.

First, the scanning electron microscope allows us to see the changes of plastic pieces with our bare eyes. However, pure observations are not enough to prove the effectiveness of our enzymes. Thus, we conducted another experiment. Though HPLC, we are able to see the enzyme and waste product curves after plastic degradation via our enzyme.

Fig.6 S121ER224QN233K Electron Scanning Microscope result
Fig.7 S121ER224QN233K HPLC 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

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.