Part:BBa_K192000

Encapsulin

Toronto 2009

BBa_K192000 is a shell-forming protein known as "encapsulin", which we derived from the TM0785 plasmid in T. maritima. The protein is a monomer which assembles into a thin icosahedral shell with a diameter of 240 angstroms resulting in a nanocompartment lined with conserved binding sites for short polypeptide tags present as C-terminal extensions of enzymes. In T. maritima, these enzymes are involved in oxidative-stress response. We have isolated the encapsulin gene in E. coli, in which we plan to characterize as an expression system for the application of channeling candidate enzyme pairs identified in silico.

Improvement from iGEM EPFL 2018

Overview

The 2018 EPFL iGEM team has improved upon this part by using a different sequence derived from a plasmid used in David Savage's lab (Cassidy-Armstutz et al., 2016). Additionally the team introduced a HexaHistidine loop (GGGGGGHHHHHHGGGGG) into the protein's sequence to improve the heat resistance and to provide better hydrodynamic properties (Moon et al., 2014). The composite part BBa_K2686005 encodes this modified Encapsulin sequence and presents an upgrade due to BsaI cut sites and sfGFP under a native promoter which allow for the rapid introduction of antigen encoding sequences to the Encapsulin monomer's C terminus.

Purification

After having tested a variety of purification procedures, heat purification at 70C for 20 minutes followed by cooling on ice for 15 minutes and a subsequent centrifugation at 12000g for 10 minutes was found to be the most efficient way of isolating the encapsulin (encapsulin ends up in supernatant).

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

Conclusion

We were unable to prove for certain that BBa_K192000 was successfully expressed in cell free and that the part assembles to a 60-mer.