Part:BBa_K2686000:Experience

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). In addition to the standard expression, a cell free expression was done with fluorescent lysine supplied in the expression environment. An SDS-PAGE was done, and fluorescent gel imaging indicated the presence of the fluorescent expressed protein at the correct size. 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).

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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