Difference between revisions of "Part:BBa K2686005"
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==Improvement upon <bbpart>BBa_K192000</bbpart>== | ==Improvement upon <bbpart>BBa_K192000</bbpart>== | ||
| − | ===Expression of <bbpart>BBa_K2686005</bbpart> derivatives=== | + | ===Expression of <bbpart>BBa_K2686005</bbpart> derivatives compared to <bbpart>BBa_K192000</bbpart>=== |
[[File:IGEM Encap.png|thumb|center|upright=3|SDS PAGE of Encapsulin proteins expressed by iGEM EPFL 2018 using a cell-free TX-TL system. Before '''(B)''' heat purification, the pellet after heat purification '''(P)''' and the supernatant after heat purification '''(S)'''. '''Triangles''' ▲ correspond to 60-mer bands of Encapsulin proteins. '''Stars''' ★ correspond to faint bands found on the lanes belonging to <bbpart>BBa_K192000</bbpart> which are likely not the multimer. '''Stars''' ✦ indicate bands belonging to the Encapsulin monomers (~32kDa). | [[File:IGEM Encap.png|thumb|center|upright=3|SDS PAGE of Encapsulin proteins expressed by iGEM EPFL 2018 using a cell-free TX-TL system. Before '''(B)''' heat purification, the pellet after heat purification '''(P)''' and the supernatant after heat purification '''(S)'''. '''Triangles''' ▲ correspond to 60-mer bands of Encapsulin proteins. '''Stars''' ★ correspond to faint bands found on the lanes belonging to <bbpart>BBa_K192000</bbpart> which are likely not the multimer. '''Stars''' ✦ indicate bands belonging to the Encapsulin monomers (~32kDa). | ||
From left to right: '''(1-3)''' Negative control of TX-TL expression system, '''(4-6)''' HexaHistidine Encapsulin <bbpart>BBa_K2686002</bbpart> showing bands ▲ belonging to the 60-mer, and in lane 6 ✦ the monomer band is visible. '''(7-8)''' are Encapsulin <bbpart>BBa_K2686001</bbpart> and ▲ Encapsulin 60-mer bands are clearly visible and in lane 8 the ✦ monomer band is visible. '''(9-11)''' shows the encapsulin from the registry <bbpart>BBa_K192000</bbpart> where no bands can be identified. '''(12)''' Ladder Precision Plus Protein™ All Blue Prestained Protein Standards #1610373. '''(13-15)''' <bbpart>BBa_K192000</bbpart> where ★ bands are present, but are unlikely to be the desired 60-mer]] | From left to right: '''(1-3)''' Negative control of TX-TL expression system, '''(4-6)''' HexaHistidine Encapsulin <bbpart>BBa_K2686002</bbpart> showing bands ▲ belonging to the 60-mer, and in lane 6 ✦ the monomer band is visible. '''(7-8)''' are Encapsulin <bbpart>BBa_K2686001</bbpart> and ▲ Encapsulin 60-mer bands are clearly visible and in lane 8 the ✦ monomer band is visible. '''(9-11)''' shows the encapsulin from the registry <bbpart>BBa_K192000</bbpart> where no bands can be identified. '''(12)''' Ladder Precision Plus Protein™ All Blue Prestained Protein Standards #1610373. '''(13-15)''' <bbpart>BBa_K192000</bbpart> where ★ bands are present, but are unlikely to be the desired 60-mer]] | ||
Revision as of 11:11, 17 October 2018
Encapsulin protein with HexaHistidine insert & sfGFP under native promoter included between BsaI cut
Composite part designed to serve as a rapid platform for fusing antigen coding sequences to the C-terminus of the encapsulin protein.
Usage and Biology
This Encapsulin protein was first modified by insertion of a hexahistidine insert to increase heat stability, and endow better hydrodynamic properties. After the encapsulin site on the plasmid (After the C-terminus), is a site for sfGFP under native promoter included within BsaI cut sites. These BsaI cut sites would allow for the rapid, scarless introduction of antigen peptides which will be expressed on the surface of the Encapsulin subunit (After the C-terminus). The insert in between the two BsaI cut sites, consisting of sfGFP with a native promoter and terminator, allows for checking the success of the insertion of the antigen (green colonies do not contain the desired peptide insert).
Improvement upon BBa_K192000
Expression of BBa_K2686005 derivatives compared to BBa_K192000
SDS PAGE of Encapsulin proteins expressed by iGEM EPFL 2018 using a cell-free TX-TL system. Before (B) heat purification, the pellet after heat purification (P) and the supernatant after heat purification (S). Triangles ▲ correspond to 60-mer bands of Encapsulin proteins. Stars ★ correspond to faint bands found on the lanes belonging to BBa_K192000 which are likely not the multimer. Stars ✦ indicate bands belonging to the Encapsulin monomers (~32kDa). From left to right: (1-3) Negative control of TX-TL expression system, (4-6) HexaHistidine Encapsulin BBa_K2686002 showing bands ▲ belonging to the 60-mer, and in lane 6 ✦ the monomer band is visible. (7-8) are Encapsulin BBa_K2686001 and ▲ Encapsulin 60-mer bands are clearly visible and in lane 8 the ✦ monomer band is visible. (9-11) shows the encapsulin from the registry BBa_K192000 where no bands can be identified. (12) Ladder Precision Plus Protein™ All Blue Prestained Protein Standards #1610373. (13-15) BBa_K192000 where ★ bands are present, but are unlikely to be the desired 60-mer
Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 77
Illegal BglII site found at 492 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 426
Illegal SapI.rc site found at 457
Illegal SapI.rc site found at 1031