Difference between revisions of "Part:BBa K1217015"

Latest revision as of 07:12, 5 October 2013

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

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

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1217015 short</partinfo>
-Bacterial microcompartments are  natural existing nano-bioreacters inside some bacteria species. They are
+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 [https://parts.igem.org/Part:BBa_K311004 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.
+[[File:Euts tagging.png|400px|thumb|none|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:HKU13_EutSHisEutS_sdspage.png|400px|thumb|left|SDS-PAGE analysis]]
+[[File:HKU13 HisEutSWestern.png|300px|thumb|none|Western Blotting analysis using anti-His antibody. It shows His tag has been fused to the EutS protein]]