Difference between revisions of "Part:BBa K3185009"
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<partinfo>BBa_K3185009 short</partinfo> | <partinfo>BBa_K3185009 short</partinfo> | ||
==Usage and Biology== | ==Usage and Biology== | ||
| − | PETase is a protein found from Ideonella sakaiensis. A paper | + | 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). |
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| − | 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. | + | 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[2]. However, we didn’t use it in our experiment. |
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==Reference== | ==Reference== | ||
| + | [1]A bacterium that degrades and assimilates poly(ethylene terephthalate) | ||
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| + | [2]Programmable polyproteins built using twin peptide superglues | ||
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===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K3185009 parameters</partinfo> | <partinfo>BBa_K3185009 parameters</partinfo> | ||
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Revision as of 10:59, 18 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 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 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
- 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
Reference
[1]A bacterium that degrades and assimilates poly(ethylene terephthalate) <bl> [2]Programmable polyproteins built using twin peptide superglues