Difference between revisions of "Part:BBa K3185000"

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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.
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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.
 
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<span class="mw-headline" id="Expression in E.coli">Expression in <i>E.coli</i></span>
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==Expression in <i>E.coli</i>==
 
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Revision as of 12:53, 17 October 2019


SPYtag inserted Tm Encapsulin

Usage and Biology

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.

Expression in E.coli


Protein Purification

Assays

Assay

Assay

Assay

Assay

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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