Part:BBa_K5236007

IsPETase W159HF229Y

This basic part encodes mutated IsPETase W159HF229Y, which comes from academic essays but is constructed by us in Escherichia coli.

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 W159HF229Y 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 W159HF229Y 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.

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.3 Mutated IsPETase Dynamic Curve
Fig.4 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.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 864
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 864
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 864
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 864
  Illegal AgeI site found at 627
  
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

Meng, Xiangxi, et al. “Protein Engineering of Stable ISPETASE for PET Plastic Degradation by Premuse.” International Journal of Biological Macromolecules, Elsevier, 19 Mar. 2021, www.sciencedirect.com/science/article/pii/S0141813021005730.