Part:BBa_K2686000:Experience

The part was designed and constructed in a pET14 vector. 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. 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 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). 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. Immunostaining and FACS were thereafter performed (Check results on iGEM EPFL 2018 page).

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