Difference between revisions of "Part:BBa K3185009"
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<partinfo>BBa_K3185009 short</partinfo>
 
<partinfo>BBa_K3185009 short</partinfo>
 
==Usage and Biology==
 
==Usage and Biology==
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PETase is a protein found from Ideonella sakaiensis.  A paper (A bacterium that degrades and assimilates poly(ethylene terephthalate)) says that PETase has PET degradation activity in a natural environment. iGEM also treats it as a useful part (BBa_K2010000).
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<br>
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We used PETase as PET binding domain because of its degradation activity. We put SpyCatcher on the N-terminus of PETase because we used SpyTag/SpyCatcher system to bind it to other parts. Also, this has three tag and cleavage sites. First is 6×His-tag inserted in the N-terminus of SpyC for protein purification. Second is MYC-tag inserted between SpyC and CBM 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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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 PETase by using SDS-PAGE.
  
 
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<partinfo>BBa_K3185009 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3185009 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>
 
<!-- Uncomment this to enable Functional Parameter display  
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K3185009 parameters</partinfo>
 
<partinfo>BBa_K3185009 parameters</partinfo>
 
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Revision as of 13:59, 17 October 2019


SPYCatcher -> PETase

Usage and Biology

PETase is a protein found from Ideonella sakaiensis. A paper (A bacterium that degrades and assimilates poly(ethylene terephthalate)) says that PETase has PET degradation activity in a natural environment. iGEM also treats it as a useful part (BBa_K2010000).

We used PETase as PET binding domain because of its degradation activity. We put SpyCatcher on the N-terminus of PETase because we used SpyTag/SpyCatcher system to bind it to other parts. Also, this has three tag and cleavage sites. First is 6×His-tag inserted in the N-terminus of SpyC for protein purification. Second is MYC-tag inserted between SpyC and CBM 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 PETase 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