Part:BBa_K3185009
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 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[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.
SDS-PAGE
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.
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