Difference between revisions of "Part:BBa K3185010"

Revision as of 11:49, 18 October 2019

SPYCatcher -> engineered PETase

Usage and Biology

Engineered PETase is a protein from Ideonella sakaiensis. The paper tries to improve the binding activity and the degradation activity of PET[1].

This time, we used engineered PETase which shows a higher binding affinity to PET than PETase in order to compare them. We put SpyCatcher on N-terminus of PETase because we used SpyTag/SpyCatcher system to bind it to other parts. It has three tags and a cleavage site. First is 6×His-tag inserted in the N-terminus of SpyCather for protein purification. Second is MYC-tag inserted between SpyCatcher and CBM (Cellose binding module [[[[[[[[[??more detail??]]]]]]]]] ) 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 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 EngiPETase 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

References

[1]Characterization and engineering of a plastic-degrading aromatic polyesterase
[2]Programmable polyproteins built using twin peptide superglues