Generating a library of mutant enzymes to degrade plastic

As plastic pollution accumulates in the ocean, it becomes a growing threat to marine and human health. Ideonella sakaiensis is a recently discovered bacterium that digests polyethylene terephthalate (PET), the plastic primarily found in single-use water bottles. This bacterium secretes two enzymes, PETase and MHETase, to digest PET and use it as a source of carbon and energy. The process by which PETase and MHETase digest PET is currently too inefficient to be useful for pollution clean-up. The goal of my project was to generate a library of mutant PETase enzymes in order to create a variant of PETase capable of digesting plastic at a rate rapid enough for practical use. To generate this library, we used error-prone PCR, in which Taq and Mutazyme polymerases introduce random mutations while replicating the PETase gene. Individual sequences from the PCR product were obtained through molecular cloning using a 2.1-TOPO vector system and competent DH5alpha E. coli, followed by Sanger sequencing. Sequencing confirmed that error-prone PCR successfully introduced mutations into the PETase gene. Furthermore, successive rounds of error-prone PCR caused an accumulation of mutations within the gene. These mutant PETases will be tested for efficiency in PET digestion in a future study.

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