Difference between revisions of "Part:BBa K2982003"
Line 7: Line 7:
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
 +
A coding sequence of the PETase double mutant W159H/S214H.
 +
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]
 +
<h1>Origin and biology</h1>
 +
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.
 +
<h1>Design</h1>
 +
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 site, W159 locate in substrate binding site, subsite II where three MHET moieties are bound through hydrophobic interaction.
 +
 +
Trp 159 in PETase has effect of extending the hydrophobic surface adjacent to the active site. Mutation, W159H narrows the binding center. The mutation site, S214 locate in the edge of the substrate binding site have effect on substrate binding.
 +
Histidine is often found in the active sites of enzymes, where its imidazole ring can readily switch between uncharged or positively charged to catalyze the making and breaking of bonds.
 +
 +
In TfCut2, Histidine 169 and Histidine 224 are located at the corresponding positions of Trpytophan 159 and Serine 214 in subsite II of IsPETase.
 +
 +
The resulting double mutant may have effect on substrate binding.
 +
<h1>Characterisation</h1>
 +
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.
 +
https://2019.igem.org/wiki/images/4/48/T--HK_GTC--48.jpg
 +
Figure 1: SDS-PAGE of purified W159H/S214H double mutant of 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.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.
 +
https://2019.igem.org/wiki/images/0/0b/T--HK_GTC--50.jpg
 +
https://2019.igem.org/wiki/images/5/54/T--HK_GTC--49.jpg
 +
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
  
 
<!-- -->
 
<!-- -->

Revision as of 14:08, 21 October 2019


Coding sequence for W159H/S214H IsPETase double mutant

A coding sequence for mutated W159H/S214H PETase from Ideonella sakainesis. It is codon optimized for Escherichia coli.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 348
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 304
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 348
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 348
    Illegal AgeI site found at 627
  • 1000
    COMPATIBLE WITH RFC[1000]