Difference between revisions of "Part:BBa K3185000"

Revision as of 12:46, 17 October 2019

SPYtag inserted Tm Encapsulin

TmEncapsulin is a protein found from Thermotoga maritima. A paper (Structural basis of enzyme encapsulation into a bacterial nano compartment) says that it consists of 60 monomers and forms capsule, Virus-like particle(VLP). 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. (Programmable polyproteins built using twin peptide superglues)

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 77
Illegal BglII site found at 597
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 426
Illegal SapI.rc site found at 457

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 <partinfo>BBa_K3185000 short</partinfo>
-aaaaa
+TmEncapsulin is a protein found from Thermotoga maritima.  A paper (Structural basis of enzyme encapsulation into a bacterial nano compartment) says that it consists of 60 monomers and forms capsule, Virus-like particle(VLP).  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. (Programmable polyproteins built using twin peptide superglues)
+ 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.
 <!-- Add more about the biology of this part here