Difference between revisions of "Part:BBa K3185009"

Revision as of 14:06, 17 October 2019

SPYCatcher -> PETase

Usage and Biology

PETase is a protein found from Ideonella sakaiensis. A paper (A bacterium that degrades and assimilates poly(ethylene terephthalate)) says that PETase has PET degradation activity in a natural environment. 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 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. (Programmable polyproteins built using twin peptide superglues) However, we didn’t use it in our experiment.

We put it between the BamHI site and the Ndel site on pET11-a. We used BL21 (DE3) for gene expression. We used the Ni-NTA Agarose for purification. After that, we confirmed the molecular weight of PETase by using SDS-PAGE.

Sequence and Features

Purification

Expression

• Cells were grown in 200ml LB media (100μg/ml Ampicillin) at 37^oC 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

Reference