Coding

Part:BBa_K3787003

Designed by: Leung Gabriel, Tam Ching Yeung, Wong Chun Hei   Group: iGEM21_HK_GTC   (2021-09-29)
Revision as of 14:57, 19 October 2021 by Ian Tam GTEYC HK (Talk | contribs)


I.s. MHETase

A coding sequence of the MHETase from Ideonella sakaiensis.

The sequence is optimized for the expression of Escherichia coli, strains DH5α and C41(DE3). It can be added to the reaction with PETase to synergize PET degradation. It can also be used as a control to compare the hydrolytic activity of PETase-MHETase chimeric proteins in the two-enzyme system.


Origin and biology

The enzyme is a hydrolase which degrades mono-(2-hydroxyethyl)terephthalic acid into monomers: terephthalic acid (TPA) and ethylene glycol (EG) 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 experiment, this gene was inserted into an expression vector, PET-21b which is used due to its high copy number, 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 E. coli C41(DE3), which we use to perform the protein induction and purification. After the protein was induced using 0.5mM IPTG, it was purified and extracted using a column with nickel resin due to the presence of a 6X His-Tag which was fused with C terminus of MHETase. After purification, SDS-PAGE and Western blot were performed to confirm the identity of expressed protein using His-Tag antibody.

T--HK_GTC--MHETase_Part_Registry.png



Our SDS-PAGE results showed that the purified MHETase protein was expressed (Figure 1, lane 3). Clear bands with correct sizes were observed in the western blot of purified MHETase proteins (Figure 2, lane 3).



These results demonstrated that our protein, MHETase, was successfully expressed and purified.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 983
    Illegal PstI site found at 1012
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 132
    Illegal PstI site found at 983
    Illegal PstI site found at 1012
    Illegal NotI site found at 680
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 983
    Illegal PstI site found at 1012
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 983
    Illegal PstI site found at 1012
    Illegal NgoMIV site found at 607
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
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