Part:BBa_K2982000
Coding sequence for wild type IsPETase
A coding sequence for wild type PETase from Ideonella sakainesis. It is codon optimized for Escherichia coli. Can serve as a standard for other experiment related to PETase.
BBa_K2982000 (Wild Type)
A coding sequence of the PETase.
This sequence is codon optimized for Escherichia coli, obtained from previous studies done on PETase.[1] It can serve as a standard for other experiment related to PETase, such as activity measurement.
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.
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 PETase.
After protein purification, an enzyme assay was performed to confirm the protein activity. The substrate used is p-nitrophenyl dodecanoate, as the ester bond is similar to that in PETase. The product, p-nitrophenol has a yellow colour. Therefore, activity can be confirmed by measuring optical density at 415nm.
As shown, there is a clear increase in optical density, confirming enzyme activity.
[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
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 348
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 304
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 348
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 348
Illegal AgeI site found at 627 - 1000COMPATIBLE WITH RFC[1000]
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