Difference between revisions of "Part:BBa K3185009"

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<partinfo>BBa_K3185009 short</partinfo>
 
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==Usage and Biology==
 
==Usage and Biology==
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PETase is a protein found from Ideonella sakaiensis.  A paper says that PETase has PET degradation activity in a natural environment[1]. iGEM also treats it as a useful part (''<partinfo>BBa_K2010000</partinfo>'').
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PETase is a protein found from Ideonella sakaiensis.  A paper says that PETase has PET degradation activity in a natural environment [1]. iGEM also treats it as a useful part (''<partinfo>BBa_K2010000</partinfo>'').
 
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Revision as of 10:56, 21 October 2019


SPYCatcher -> PETase

Usage and Biology

PETase is a protein found from Ideonella sakaiensis. A paper says that PETase has PET degradation activity in a natural environment [1]. 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(BBa_K1159200) on the N-terminus of PETase because we used SpyCatcher/SpyTag 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[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

Fig.1 SDS-PAGE of imidazole elutes, CBB stained


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, we didn't see any band in the specific molecular weight of the protein.

Result

Reference

1 Yang, Y., Yang, J., and Jiang, L. (2016).
Comment on "a bacterium that degrades and assimilates poly(ethylene terephthalate) ".
Science (80-. ). 353, 759.

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.