Difference between revisions of "Part:BBa K2686005"

Latest revision as of 01:55, 18 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 derived from K2686002 has a hexahistidine insert to increase heat stability, and endow better hydrodynamic properties (Moon et al., 2014). 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).

In our case this part was used in order to build K2686000 using a OT1 peptide coding sequence (Choi et al., 2016).

Improvement upon BBa_K192000

Expression of BBa_K2686005 precursors 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

References

Choi, B., Moon, H., Hong, S., Shin, C., Do, Y., Ryu, S. and Kang, S. (2016). Effective Delivery of Antigen–Encapsulin Nanoparticle Fusions to Dendritic Cells Leads to Antigen-Specific Cytotoxic T Cell Activation and Tumor Rejection. ACS Nano, 10(8), pp.7339-7350.

Moon, H., Lee, J., Min, J. and Kang, S. (2014). Developing Genetically Engineered Encapsulin Protein Cage Nanoparticles as a Targeted Delivery Nanoplatform. Biomacromolecules, 15(10), pp.3794-3801.

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 492
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
Illegal SapI.rc site found at 1031

@@ Line 8: / Line 8: @@
 ===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).
+This Encapsulin protein derived from <bbpart>K2686002</bbpart> has a hexahistidine insert to increase heat stability, and endow better hydrodynamic properties (Moon et al., 2014). 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).
+In our case this part was used in order to build <bbpart>K2686000</bbpart> using a OT1 peptide coding sequence (Choi et al., 2016).
+==Improvement upon <bbpart>BBa_K192000</bbpart>==
+===Expression of <bbpart>BBa_K2686005</bbpart> precursors 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''' &#9650; correspond to 60-mer bands of Encapsulin proteins. '''Stars''' &#9733; correspond to faint bands found on the lanes belonging to <bbpart>BBa_K192000</bbpart> which are likely not the multimer. '''Stars''' &#10022; 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 &#9650; belonging to the 60-mer, and in lane 6 &#10022; the monomer band is visible. '''(7-8)''' are Encapsulin <bbpart>BBa_K2686001</bbpart> and &#9650; Encapsulin 60-mer bands are clearly visible and in lane 8 the &#10022; 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 &#9733; bands are present, but are unlikely to be the desired 60-mer]]
+===References===
+Choi, B., Moon, H., Hong, S., Shin, C., Do, Y., Ryu, S. and Kang, S. (2016). Effective Delivery of Antigen–Encapsulin Nanoparticle Fusions to Dendritic Cells Leads to Antigen-Specific Cytotoxic T Cell Activation and Tumor Rejection. ACS Nano, 10(8), pp.7339-7350.
+Moon, H., Lee, J., Min, J. and Kang, S. (2014). Developing Genetically Engineered Encapsulin Protein Cage Nanoparticles as a Targeted Delivery Nanoplatform. Biomacromolecules, 15(10), pp.3794-3801.
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