Difference between revisions of "Part:BBa K3185009"

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<h3><font size="4.5">Purification </font></h3>
 
<h3><font size="4.5">Purification </font></h3>
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1. <i>E.coli<i/> which expressed this part were lysed with sonification.<br>
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1. <i>E.coli</i> which expressed this part were lysed with sonification.<br>
 
2. Proteins are purified from lysate with Ni-NTA agarose(QIAGEN).<br>
 
2. Proteins are purified from lysate with Ni-NTA agarose(QIAGEN).<br>
 
3. Imidazole eluates were visualized and confirmed by SDS-PAGE followed by CBB staining.<br>
 
3. Imidazole eluates were visualized and confirmed by SDS-PAGE followed by CBB staining.<br>

Revision as of 13:28, 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

We couldn't get any results in our experiment because of failure of protein purification.

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.