Difference between revisions of "Part:BBa K3185009"
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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> | ||
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| − | This purification method failed. As shown in Fig.1, we didn't see any band in the specific molecular weight of the protein. | + | This purification method failed. As shown in Fig.1, we didn't see any band in the specific molecular weight of the protein. |
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==Result== | ==Result== | ||
Revision as of 15:55, 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 751
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1318
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1074
- 1000COMPATIBLE 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, we didn't see any band in the specific molecular weight of the protein.
Result
We couldn't get any result in our experiment because we failed the 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.