Composite

Part:BBa_K1217015

Designed by: Ma Tsz Shan   Group: iGEM13_Hong_Kong_HKU   (2013-09-15)
Revision as of 07:10, 5 October 2013 by ShannonMa (Talk | contribs)

Native Eut Microcompartment with Surface His-tag display

Bacterial microcompartments (MCP)are natural existing nano-bioreacters inside some bacteria species. They are Protein-based polyhedral microcompartments which ranges from 20-200 nm in diameter. Despite its versality is side, their main function is enhance unfavoured metabolic reaction by sequestering functionally related enzymes, regulating substrate and small metabolites access. Eut microcompartment BBa_K311004 is one example which has been established by [http://2010.igem.org/Team:Minnesota/Project Team Minnesota 2010]

Different kinds of protein cage with various size have been engineered as target specific nano-containers: e.g.Viral capsids, ferritins & heat shock proteins. One important feature to enable the use of target-specific nano-container is the editable nature of protein cage's surface. We investigated the possibility to display specific peptide on the Eut microcompartment surface.

We find, among the 5 structural genes for the assembly of Eut MCP, EutS is suitable for surface peptide display.

EutS monomer N terminus is extruding out of the EutS hexamer’s surface.Studies showed that convex side of the EutS hexamer is the exterior side of Eut MCP.

Hence we hypothisezed that we can present designed targeting peptide on the exterior surface of the MCP by fusing the peptide to the EutS N terminus. And the additional peptide should pose little steric effect on the MCP assembly. As a proof of principle, we fused a 6 histidine tag to the EutS gene. We hope we can purify the MCP easily using the nickel affinity column.

Addition of His tag to EutS protein

File:EutSHisEutS sdspage.png
SDS-PAGE analysis
Western Blotting analysis using anti-His antibody. It shows His tag has been fused to the EutS protein



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 411
    Illegal NotI site found at 1321
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 624
    Illegal BglII site found at 1534
    Illegal XhoI site found at 419
    Illegal XhoI site found at 1329
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1248
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 836


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