Difference between revisions of "Part:BBa K3185000"
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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 C-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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We used TmEncapsulin as a biological polymer. We put Spytag inside TmEncapsulin because we used SpyTag/SpyCatcher system to bind it to other parts[2]. Also, this has three tag and cleavage sites. First is 6×His-tag inserted in the C-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 inserted between the C-terminus of Encapsulin and 6x-His-tag because, in a paper, it is said that 6x-His-tag inserted in the C-terminus of Encapsulin is not presented on the surface of Encapsulin well, so it can’t bind to Ni-NTA Agarose beads. In the same paper, it is also said that heat-resistance is improved when inserting 6x-His-tag and linker between #43 and #44 amino acids of native encapsulin, so we designed it like that[3].  
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<span class='h3bb'>Sequence and Features</span>
 
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==Purification==
 
==Purification==
 
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Revision as of 11:57, 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[2]. Also, this has three tag and cleavage sites. First is 6×His-tag inserted in the C-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 inserted between the C-terminus of Encapsulin and 6x-His-tag because, in a paper, it is said that 6x-His-tag inserted in the C-terminus of Encapsulin is not presented on the surface of Encapsulin well, so it can’t bind to Ni-NTA Agarose beads. In the same paper, it is also said that heat-resistance is improved when inserting 6x-His-tag and linker between #43 and #44 amino acids of native encapsulin, so we designed it like that[3].



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

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