Difference between revisions of "Part:BBa K3185010"

 
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<partinfo>BBa_K3185010 short</partinfo>
 
<partinfo>BBa_K3185010 short</partinfo>
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==Usage and Biology==
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Engineered PETase is a protein from Ideonella sakaiensis. The paper (Characterization and engineering of a plastic-degrading aromatic polyesterase) tries to improve the binding activity and the degradation activity of PET.
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<br>
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<br>
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This time, we used engineered PETase which shows a higher binding affinity to PET than PETase in order to compare them. We put SpyCatcher on N-terminus of PETase because we used SpyTag/SpyCatcher system to bind it to other parts.
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It has three tags and a cleavage site. First is 6×His-tag inserted in the N-terminus of SpyCather for protein purification. Second is MYC-tag inserted between SpyCatcher and CBM (Cellose binding module ??more detail??) to detect it by using the antibody. Third is a TEV protease site because, in the paper, it was used for protein purification. (Programmable polyproteins built using twin peptide superglues) However, we didn’t use it in our experiment.
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<br>
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<br>
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We put it between the BamHI site and the Ndel site on pET11-a. We used BL21 (DE3) for gene expression. We used the Ni-NTA  agarose for purification. After that, we confirmed the molecular weight of EngiPETase by using SDS-PAGE.
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3185010 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3185010 SequenceAndFeatures</partinfo>
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==Purification==
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<br>
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<h3><font size="4.5">Expression</font> </h3>
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<ul>
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<li>Cells were grown in 200ml LB media (100μg/ml Ampicillin) at 37<sup>o</sup>C shaking at 140 rpm to an OD600 of 0.5, verifying via a spectrophotometer.
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</ul>
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<ul>
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<li>Protein was expressed in 0.1mM IPTG for 2hours.
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</ul>
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<h3><font size="4.5">SDS-PAGE</font></h3>
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<br>
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<br>
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K3185010 parameters</partinfo>
 
<partinfo>BBa_K3185010 parameters</partinfo>
 
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Revision as of 14:01, 17 October 2019


SPYCatcher -> engineered PETase

Usage and Biology

Engineered PETase is a protein from Ideonella sakaiensis. The paper (Characterization and engineering of a plastic-degrading aromatic polyesterase) tries to improve the binding activity and the degradation activity of PET.

This time, we used engineered PETase which shows a higher binding affinity to PET than PETase in order to compare them. We put SpyCatcher on N-terminus of PETase because we used SpyTag/SpyCatcher system to bind it to other parts. It has three tags and a cleavage site. First is 6×His-tag inserted in the N-terminus of SpyCather for protein purification. Second is MYC-tag inserted between SpyCatcher and CBM (Cellose binding module ??more detail??) to detect it by using the antibody. Third is a TEV protease site because, in the paper, it was used for protein purification. (Programmable polyproteins built using twin peptide superglues) However, we didn’t use it in our experiment.

We put it between the BamHI site and the Ndel site on pET11-a. We used BL21 (DE3) for gene expression. We used the Ni-NTA agarose for purification. After that, we confirmed the molecular weight of EngiPETase by using SDS-PAGE.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 751
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1318
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1074
  • 1000
    COMPATIBLE WITH RFC[1000]

Purification


Expression

  • Cells were grown in 200ml LB media (100μg/ml Ampicillin) at 37oC shaking at 140 rpm to an OD600 of 0.5, verifying via a spectrophotometer.
  • Protein was expressed in 0.1mM IPTG for 2hours.

SDS-PAGE