Difference between revisions of "Part:BBa K3039001"

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PETase T88A_S121E_D186H_R280A

The enzymes PETase and MHETase were first discovered in Ideonella sakaiensis in 2016 by a group of researchers in Japan. These enzymes were found to degrade polyethylene terephthalate (PET) into its monomers, terephthalic acid (TPA) and ethylene glycol (EG). PETase degrades PET into Mono-(2-hydroxyethyl)terephthalic acid (MHET), Bis(2-Hydroxyethyl) terephthalate (BHET) and TPA, the main product being MHET. MHET is further degraded by MHETase into TPA and EG. We are aiming to use mutants of these enzymes to degrade the microfibres that are coming off clothing during washing cycles. The enzymes would be secreted into a filter that captures the microfibres. Different mutations that have been reported in past papers to increase the activity of PETase have been combined into a novel mutant, in order to test if this would result in an overly active mutant. This sequence is the Escherichia coli K12 (E. coli K12) codon optimized DNA of the novel mutant of PETase, with an attached His tag. The His tag was attached in order to more easily identify the enzymes.

The native predicted signal peptide (Met1-Ala33) was removed from the WT PETase sequence (Seo et al 2019) and replaced with a start codon (Met), however all mutations are numbered according to the full-length WT sequence. The amino acid sequence was submitted to Twist Bioscience who codon optimised the sequence for E. coli, ensuring that there were no forbidden restriction sites, BsaI or SapI, to allow for potential TypeIIS assembly. The resulting CDS was synthesised and cloned, by Twist, into pET28. This added a 63 AA His-tag and thrombin cleavage site to the N-terminal of the protein, a T7 promoter and T7 terminator.

Western blot of the soluble fraction of Arctic Express strain showing expression of all mutants. The PageRuler Plus prestained protein ladder was used and labeled with the corresponding sizes. The negative control is labeled with 1. This part (PETase T88A_S121E_D186H_R280A) is labeled with 2. A clear band is visible with a size of about 30 kDa which is the size of PETase with the His tag attached to it.

Western blot of the soluble fraction of Rosetta Gami strain showing expression of all mutants. The PageRuler Plus prestained protein ladder was used and labeled with the corresponding sizes. The negative control is labeled with 1. This part (PETase T88A_S121E_D186H_R280A) is labeled with 2. A clear band is visible with a size of about 30 kDa which is the size of PETase with the His tag attached to it.

Expression in E.coli

Protein Purification

Assays

Esterase Assays

Thermal Stability Assay

Thermal Shift Assay

PET Assay