Part:BBa_K2982004
Coding sequence for W159H/S245I IsPETase double mutant
A coding sequence for mutated W159H/S245I PETase from Ideonella sakainesis. It is codon optimized for Escherichia coli.
Usage and Biology
A coding sequence of the PETase double mutant W159H/S245I.
This sequence is modified from a sequence for wild type PETase which was also codon optimised for Escherichia coli, obtained from previous studies done on 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.
Design
We analyze the rationale for PETase mutant design from previous studies done on the residue modification of this enzyme. A clear trend in most successful mutation attempts is that an increase in hydrophobicity or a binding site similar to T. fusca cutinase, which is narrower.
The mutation sites locate in substrate binding site, subsite II where three MHET moieties are bound through hydrophobic interaction.
In TfCut2, Histidine 169 residues and Isoleucine 253 are located at the corresponding positions of Trpytophan 159 and Serine 245 in subsite II of IsPETase. The resulting double mutant makes the substrate binding site, substrate II more cutinase-like and increases the hydrophobic property of the enzyme.
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 0.05mM 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.
Figure 1: SDS-PAGE of purified W159H/S245I PETase. A band of around 30 kDa is clearly shown
As shown above, the thick band around 30 kDa shows successful expression of the construct.
After protein purification, an enzyme assay is performed to confirm the protein activity.
Improvement (Of BBa_K2010000)
The part BBa_K2010000 from Team iGEM16_Harvard_BioDesign coded for the wild type of PETase from Ideonella sakaiensis. While the wild type has significant activity for the degradation of PETase, it is not high enough for applications.
To improve the activity of wild type PETase, we used site-directed mutagenesis to make a double mutant, and W159H/S245I is the one which shows the largest improvement from the wild type. (For details of our design, check the “Design” section above)
Then,we tested the mutant using an enzyme assay with p-nitrophenyl dodecanoate as the substrate. 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 W159H/S245I and Wild Type PETase. It can be seen that W159H/S245I has a higher activity
As shown in the data, the W159H/S245I double mutant exhibits 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 of the wild type, and thus, the part is an improvement of BBa_K2010000.
[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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