Latest revision as of 14:39, 21 October 2019

Coding sequence for W159H/S238F IsPETase double mutant

A coding sequence for mutated W159H/S238F PETase from Ideonella sakainesis. It is codon optimized for Escherichia coli.

A coding sequence of the PETase double mutant W159H/S238F.

This sequence is codon optimized for Escherichia coli, obtained from a previous study done on this mutant of PETase. [1]

Origin and biology

The enzyme is a hydrolase which degrades polyethylene terephthalate into simple molecules: MHET, BHET, and TPA by cleavage of the ester bond within the polymer. It was originally found in the bacteria Ideonella sakaiensis, which uses PET as a carbon source, and integrates the degradation products into its metabolic cycle.

This double mutant is designed from a previous study done on mutants of the enzyme. It was a successful mutant and can be served as a standard for our own mutants.

Characterisation

In our experiments, to insert this gene into cells, the PET-21b vector is used due to its high copy number and the presence of T7 promoter and a lac operon. We use DH5ɑ as host cells due to its high insert stability. Then, extracted DNA is transformed into C41(DE3) cells, which we use to perform the protein induction due to the toxic nature of PETase.

After the protein is induced using 0.5mM IPTG, it can be purified and extracted using a column with nickel resin due to a 6X His-Tag fused with PETase outside the globular structure. After purification, SDS-PAGE can be performed to confirm successful expression.

As shown above, the thick band around 30 kDa shows successful expression of the construct.

After protein purification, an enzyme assay can be performed to confirm the protein activity.

Validation

As shown in literature, the PETase mutant which BBa_K2982001 codes for has a higher activity than wild type PETase, encoded by BBa_K2982000.[1] We test the mutant using an enzyme assay with p-nitrophenyl dodecanoate as the substrate. When the ester bond of the substrate is cut by the PETase, it produces p-nitrophenol which has a yellow colour. Therefore, the optical density at 415nm should increase. By comparing the optical densities of the reaction mixture with the mutant and the wild type PETase, we can see how the mutation affects activity.

Figure 2: Optical densities at 415nm for reaction mixtures with wild type and W159H/S238F PETase. It can be seen that W159H/S238F has a higher OD at all times.

Figure 3: Percentage increases of optical density for reaction mixtures with W159H/S238F PETase. It can be seen that W159H/S238F has a higher percentage increase at all times.

As shown in the data, the W159H/S238F double mutant has a higher rate of percentage increase, and also higher overall increase in optical density at 415nm at all times. This shows that the activity of the mutant is undeniably higher than that of the wild type. This verifies that the mutant is an enhancement version of the wild type.

[1]:Austin, H. P., Allen, M. D., Donohoe, B. S., Rorrer, N. A., Kearns, F. L., Silveira, R. L., . . . Beckham, G. T. (2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19). doi:10.1073/pnas.1718804115

Usage and Biology

Sequence and Features