Difference between revisions of "Part:BBa K3787002"
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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 6× 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. | 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 6× 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. | ||
| − | <center>https://2021.igem.org/wiki/images/2/25/T--HK_GTC | + | <center>https://2021.igem.org/wiki/images/2/25/T--HK_GTC--27_28.png<center> |
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). <br><br> | 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). <br><br> | ||
Revision as of 15:06, 19 October 2021
S245I PETase-MHETase
A coding sequence of the S245I PETase-MHETase chimera.
The sequence is optimized for the expression of Escherichia coli, strains DH5α and C41(DE3).
Origin and Biology
This part is a chimeric protein used to depolymerize PET into its constituting monomers. It consists of two enzymes, S245I PETase mutant and MHETase, both belong to the hydrolase superfamily. The PETase part degrades PET into bis(2-hydroxyethyl) terephthalic acid (BHET), mono(2-hydroxyethyl) terephthalic acid (MHET), and terephthalic acid (TPA), while the MHETase part degrades MHET into TPA and ethylene glycol (EG) by cleavage of the ester bond within the polymer.
The C-terminus of PETase is linked to the N-terminus of MHETase using a 12 amino acid serine-glycine linker.
These two enzymes were originally found in the bacteria Ideonella sakaiensis, which uses PET as a carbon source, and integrates the degradation products into its metabolic cycle.
Design of S245I PETase mutant
The mutant makes the substrate binding site, subunit II more cutinase-like and increases the hydrophobic property of the enzyme. It was created by our team in iGEM 2019.
Link: https://parts.igem.org/wiki/index.php?title=Part:BBa_K2982005
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 6× 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.
<center>
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
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 348
Illegal PstI site found at 1889
Illegal PstI site found at 1918 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 304
Illegal NheI site found at 1038
Illegal PstI site found at 1889
Illegal PstI site found at 1918
Illegal NotI site found at 1586 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 348
Illegal PstI site found at 1889
Illegal PstI site found at 1918 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 348
Illegal PstI site found at 1889
Illegal PstI site found at 1918
Illegal NgoMIV site found at 1513
Illegal AgeI site found at 627 - 1000COMPATIBLE WITH RFC[1000]
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