Difference between revisions of "Part:BBa K3185000"
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<partinfo>BBa_K3185000 short</partinfo> | <partinfo>BBa_K3185000 short</partinfo> | ||
==Usage and Biology== | ==Usage and Biology== | ||
− | TmEncapsulin is a protein found from <i>Thermotoga maritima</i>. A paper | + | TmEncapsulin is a protein found from <i>Thermotoga maritima</i>. A paper says that it consists of 60 monomers and forms capsule, Virus-like particle(VLP)[1]. iGEM also treats it as a useful part (BBa_K192000). |
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− | We used TmEncapsulin as a biological polymer. We put Spytag inside TmEncapsulin 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 TmEncapsulin for protein purification. Second is HA-tag inserted between TmEncapsulin and 6x-His-tag to detect it by using antibodies. Third is a 6x-His tag because, in the paper, it was used for improving the heat-resistant ability of TmEncapsulin. | + | We used TmEncapsulin as a biological polymer. We put Spytag inside TmEncapsulin 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 TmEncapsulin for protein purification. Second is HA-tag inserted between TmEncapsulin and 6x-His-tag to detect it by using antibodies. Third is a 6x-His tag because, in the paper, it was used for improving the heat-resistant ability of TmEncapsulin.[2] |
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==References== | ==References== | ||
+ | [1]Structural basis of enzyme encapsulation into a bacterial nano compartment | ||
+ | [2]Programmable polyproteins built using twin peptide superglues | ||
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===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K3185000 parameters</partinfo> | <partinfo>BBa_K3185000 parameters</partinfo> | ||
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Revision as of 06:01, 18 October 2019
SPYtag inserted Tm Encapsulin
Usage and Biology
TmEncapsulin is a protein found from Thermotoga maritima. A paper says that it consists of 60 monomers and forms capsule, Virus-like particle(VLP)[1]. iGEM also treats it as a useful part (BBa_K192000).
We used TmEncapsulin as a biological polymer. We put Spytag inside TmEncapsulin 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 TmEncapsulin for protein purification. Second is HA-tag inserted between TmEncapsulin and 6x-His-tag to detect it by using antibodies. Third is a 6x-His tag because, in the paper, it was used for improving the heat-resistant ability of TmEncapsulin.[2]
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 SpyCatcher inserted TmEncapusulin by using SDS-PAGE.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 77
Illegal BglII site found at 597 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 426
Illegal SapI.rc site found at 457
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
References
[1]Structural basis of enzyme encapsulation into a bacterial nano compartment [2]Programmable polyproteins built using twin peptide superglues