Difference between revisions of "Part:BBa K3185010"
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<partinfo>BBa_K3185010 short</partinfo>
 
<partinfo>BBa_K3185010 short</partinfo>
 
==Usage and Biology==
 
==Usage and Biology==
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Engineered PETase is a protein from Ideonella sakaiensis. The paper tries to improve the binding activity and the degradation activity of PET[1].
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Engineered PETase is a protein from Ideonella sakaiensis. The paper tries to improve the binding activity and the degradation activity of PET [1].
 
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We used engineered PETase which shows a higher binding affinity to PET than PETase in order to compare them. We put SpyCatcher(''<partinfo>BBa_K1159200</Partinfo>'') on N-terminus of PETase because we used SpyCatcher/SpyTag system to bind it to other parts.  
 
We used engineered PETase which shows a higher binding affinity to PET than PETase in order to compare them. We put SpyCatcher(''<partinfo>BBa_K1159200</Partinfo>'') on N-terminus of PETase because we used SpyCatcher/SpyTag 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 PETase to detect it by using the antibody. Third is a TEV protease site because, in the paper, it was used for protein purification[2]. However, we didn’t use it in our experiment.  
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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 PETase to detect it by using the antibody. Third is a TEV protease site because, in the paper, it was used for protein purification [2]. However, we didn’t use it in our experiment.  
 
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==Purification==
 
==Purification==
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[[File:engineered PETase.png|300px|thumb|left|♪]]
 
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<h3><font size="4.5">Expression</font> </h3>
 
<h3><font size="4.5">Expression</font> </h3>
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<li>Protein was expressed in 0.1mM IPTG for 2hours.
 
<li>Protein was expressed in 0.1mM IPTG for 2hours.
 
</ul>
 
</ul>
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<h3><font size="4.5">SDS-PAGE</font></h3>
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<h3><font size="4.5">Purification </font></h3>
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[[File:engineered PETase.png|800px|thumb|left|♪]]
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1. E.coli which expressed this part were lysed with sonification.<br>
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2. Proteins are purified from lysate with Ni-NTA agarose(QIAGEN).<br>
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3. Imidazole eluates were visualized and confirmed by SDS-PAGE followed by CBB staining.<br>
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<br>
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This purification method failed. As shown in Fig.1, the protein successfully purified.
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Revision as of 09:42, 21 October 2019


SPYCatcher -> engineered PETase

Usage and Biology

Engineered PETase is a protein from Ideonella sakaiensis. The paper tries to improve the binding activity and the degradation activity of PET [1].

We used engineered PETase which shows a higher binding affinity to PET than PETase in order to compare them. We put SpyCatcher(BBa_K1159200) on N-terminus of PETase because we used SpyCatcher/SpyTag 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 PETase to detect it by using the antibody. Third is a TEV protease site because, in the paper, it was used for protein purification [2]. However, we didn’t use it in our experiment.

We put it between BamHI site and Ndel site on pET11-a. The expression plasmids were introduced into BL21(DE3) and expressed by T7 promoter/ T7 RNAP system. Ni-NTA agarose was used for the purification.

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.

Purification

1. E.coli which expressed this part were lysed with sonification.
2. Proteins are purified from lysate with Ni-NTA agarose(QIAGEN).
3. Imidazole eluates were visualized and confirmed by SDS-PAGE followed by CBB staining.

This purification method failed. As shown in Fig.1, the protein successfully purified.











































Result

References

1 Austin, H.P., Allen, M.D., Donohoe, B.S., Rorrer, N.A., Kearns, F.L., Silveira, R.L., Pollard, B.C., Dominick, G., Duman, R., Omari, K. El, et al. (2018).
Characterization and engineering of a plastic-degrading aromatic polyesterase.
Proc. Natl. Acad. Sci. U. S. A. 115, E4350–E4357.

2 Veggiani, G., Nakamura, T., Brenner, M.D., Gayet, R. V., Yan, J., Robinson, C. V., and Howarth, M. (2016).
Programmable polyproteams built using twin peptide superglues.
Proc. Natl. Acad. Sci. U. S. A. 113, 1202–1207.