Difference between revisions of "Part:BBa K3039020"
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<span class="mw-headline" id="Expression in E.coli">Expression in <i>E.coli</i></span>
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<h3><font size="4.5"> Protein Purification</font> </h2>
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<h3><font size="4.5"> Assays</font></h3>
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<h4><u> Esterase Assays  </u></h4>
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<h4><u> Thermal Stability Assay </u> </h4>
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<h4><u> Thermal Shift Assay  </u></h4>
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<h4><u> PET Assay  </u></h4>
 
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Revision as of 17:01, 17 October 2019


PETase Reconstructed Ancestor 4

The enzymes PETase and MHETase were first discovered in Ideonella sakaiensis in 2016 by a group of researchers in Japan. These enzymes were found to degrade polyethylene terephthalate (PET) into its monomers, terephthalic acid (TPA) and ethylene glycol (EG). PETase degrades PET into Mono-(2-hydroxyethyl)terephthalic acid (MHET), Bis(2-Hydroxyethyl) terephthalate (BHET) and TPA, the main product being MHET. MHET is further degraded by MHETase into TPA and EG. We are aiming to use mutants of these enzymes to degrade the microfibres that are coming off clothing during washing cycles. The enzymes would be secreted into a filter that captures the microfibres. This is the sequence of one of the four reconstructed ancestors of PETase with a His tag attached to it. The sequence has been obtained through the method of ancestral reconstruction. The His tag has been used in order to more easily identify the enzyme.



Expression in E.coli


Protein Purification

Assays

Esterase Assays

Thermal Stability Assay

Thermal Shift Assay

PET Assay



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 433
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 433
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 433
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 433
  • 1000
    COMPATIBLE WITH RFC[1000]