Difference between revisions of "Part:BBa K2686000:Experience"
 
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The part was designed and constructed in pET vector. The sequence was then confirmed on pET vector using sanger sequencing. The part was expressed in the pET vector using a BL21 DE3 cell free expression system. After expression and purification, the part was validated in multiple ways. First, multiple SDS-PAGE experiments indicate that our expressed protein is at the correct hypothetical size. The expressed protein size (20-25 nm) was validated using Dynamic Light Scattering (DLS). The expressed part was also submitted to MassSpec (Check results on iGEM EPFL 2018 page)..
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The part was designed and constructed in the original pET14 vector from <bbpart>K2686001</bbpart> (Cassidy-Armstutz et al., 2016, Addgene plasmid #86405). The sequence was confirmed using Sanger sequencing. The part was expressed in the pET14 vector using a BL21 DE3 TX-TL (Sun et al., 2013) cell free expression system as used by 2017 iGEM EPFL. After expression and purification, the part was validated in multiple ways. First, multiple SDS-PAGE experiments indicate that our expressed protein is at the correct hypothetical size, which was further confirmed by lysine BODIPY tRNA expression (32.96kDa for the monomer and 1.98MDa for the 60-mer).
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In addition to the standard expression, a cell free expression was done with fluorescent lysine supplied in the expression environment. An SDS-PAGEwas done, and fluorescent gel imaging indicated the presence of the fluorescent expressed protein at the correct size.
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The HexaHistidine-OT1 Encapsulin construct was also expressed and presented to dendritic cells to check its ability to induce the presentation of the OT1 antigen on MHC I complexes on the dendritic cells.  
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The Encapsulin-OVA construct was also expressed and presented to dendritic cells to check its ability to induce the presentation of the OVA antigen on MHC I complexes on the dendritic cells. Immunostaining and FACS were thereafter performed (Check results on iGEM EPFL 2018 page).
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The part was then amplified out of the pET vector and inserted into the pSB1C3 plasmid backbone, and submitted. There was not enough time to perform Sanger sequencing on the obtained pSB1C3 plasmid, but colony PCR gave us the correct insert size.
 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
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how you used this part and how it worked out.
 
  
 
===Applications of BBa_K2686000===
 
===Applications of BBa_K2686000===
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===References===
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Cassidy-Amstutz, C., Oltrogge, L., Going, C., Lee, A., Teng, P., Quintanilla, D., East-Seletsky, A., Williams, E. and Savage, D. (2016). Identification of a Minimal Peptide Tag for in Vivo and in Vitro Loading of Encapsulin. Biochemistry, 55(24), pp.3461-3468.

Latest revision as of 22:27, 17 October 2018

The part was designed and constructed in the original pET14 vector from K2686001 (Cassidy-Armstutz et al., 2016, Addgene plasmid #86405). The sequence was confirmed using Sanger sequencing. The part was expressed in the pET14 vector using a BL21 DE3 TX-TL (Sun et al., 2013) cell free expression system as used by 2017 iGEM EPFL. After expression and purification, the part was validated in multiple ways. First, multiple SDS-PAGE experiments indicate that our expressed protein is at the correct hypothetical size, which was further confirmed by lysine BODIPY tRNA expression (32.96kDa for the monomer and 1.98MDa for the 60-mer). The HexaHistidine-OT1 Encapsulin construct was also expressed and presented to dendritic cells to check its ability to induce the presentation of the OT1 antigen on MHC I complexes on the dendritic cells.


Applications of BBa_K2686000

User Reviews

UNIQ2817c051ce4c4e85-partinfo-00000001-QINU UNIQ2817c051ce4c4e85-partinfo-00000002-QINU

References

Cassidy-Amstutz, C., Oltrogge, L., Going, C., Lee, A., Teng, P., Quintanilla, D., East-Seletsky, A., Williams, E. and Savage, D. (2016). Identification of a Minimal Peptide Tag for in Vivo and in Vitro Loading of Encapsulin. Biochemistry, 55(24), pp.3461-3468.