Difference between revisions of "Part:BBa K2686002"
(→‎Expression & Purification)
Line 23: Line 23:
  
 
===Expression & Purification===
 
===Expression & Purification===
 +
===Expression===
 
A cell free expression system was used to synthesize the encapsulin proteins ''in vitro''. The TX-TL cell free system is a robust way to express proteins (Sun et al., 2013), and was used by last year's EPFL iGEM team [[2017.igem.org/Team:EPFL/Description/Lysates| Aptasense]].  
 
A cell free expression system was used to synthesize the encapsulin proteins ''in vitro''. The TX-TL cell free system is a robust way to express proteins (Sun et al., 2013), and was used by last year's EPFL iGEM team [[2017.igem.org/Team:EPFL/Description/Lysates| Aptasense]].  
−
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.
+
 
 +
[[File:SDS LysBodipy.tiff|thumb|center|upright=3|SDS PAGE of encapsulins expressed in cell free TX-TL system with Lysine-BODIPY fluorescent tRNA's. The two different sets of lanes correspond to different heat denaturation temperatures (70C and 100C for 15 minutes).
 +
From left to right: '''(N)''' Negative control (cell-free PURE expression without DNA and no purification), '''(L)''' Positive control with DNA coding for Luciferase (37kDa), '''(H)''' HexaHistidine Encapsulin ([[parts.igem.org/Part:BBa_K2686002|BBa_K2686002]]) showing bands for the encapsulin multimer high on the gel lanes as well as the monomer around 31kDa, '''(R)''' Encapsulin ([[parts.igem.org/Part:BBa_K2686001|BBa_K2686001]]) without HexaHistidine linker, '''(N)''' Negative control (cell-free PURE expression without DNA and 100C denaturation), '''(Ladder)'''  LC5928 BenchMark™ Fluorescent Protein Standard, '''(L)''' Positive control with DNA coding for Luciferase (37kDa),  '''(H)''' HexaHis Encapsulin ([[parts.igem.org/Part:BBa_K2686002|BBa_K2686002]]) showing bands for the encapsulin multimer high on the gel lanes as well as the monomer around 31kDa, '''(R)''' Encapsulin ([[parts.igem.org/Part:BBa_K2686001|BBa_K2686001]]) without HexaHistidine linker, '''(N)''' Negative control (cell-free TX-TL expression without DNA and 70C denaturation).]]
  
 
===Assembly===
 
===Assembly===

Revision as of 12:33, 16 October 2018


Encapsulin protein with HexaHistidine insert

This part encodes a modified Thermotoga maritima Encapsulin protein. The part is optimized for expression in E. coli and has an additional HexaHistidine (GGGGGGHHHHHHGGGGG) insert between amino acids 43 and 44, forming a loop on the interior surface of the encapsulin monomer providing higher heat resistance and stability, and better hydrodynamic properties (Moon et al., 2014).

Usage and Biology

The part can be used to deliver cargo, both on the outer surface of the nanoparticle by fusing a peptide in between the 139/140 Amino Acids as well as the protein's C terminus. Cargo proteins can also be loaded inside the nano-cage using a tag binding to Encapsulin's interior surface (Cassidy-Amstutz et al., 2016). The protein is modified with an additional amino acid sequence (GGGGGGHHHHHHGGGGG) between positions 43/44 granting it better stability and high heat resistance (Moon et al., 2014).

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


Characterization

A variety of different characterization techniques were used to assess the properties of the encapsulin protein cage.

Expression & Purification

Expression

A cell free expression system was used to synthesize the encapsulin proteins in vitro. The TX-TL cell free system is a robust way to express proteins (Sun et al., 2013), and was used by last year's EPFL iGEM team Aptasense.

SDS PAGE of encapsulins expressed in cell free TX-TL system with Lysine-BODIPY fluorescent tRNA's. The two different sets of lanes correspond to different heat denaturation temperatures (70C and 100C for 15 minutes). From left to right: (N) Negative control (cell-free PURE expression without DNA and no purification), (L) Positive control with DNA coding for Luciferase (37kDa), (H) HexaHistidine Encapsulin (BBa_K2686002) showing bands for the encapsulin multimer high on the gel lanes as well as the monomer around 31kDa, (R) Encapsulin (BBa_K2686001) without HexaHistidine linker, (N) Negative control (cell-free PURE expression without DNA and 100C denaturation), (Ladder) LC5928 BenchMark™ Fluorescent Protein Standard, (L) Positive control with DNA coding for Luciferase (37kDa), (H) HexaHis Encapsulin (BBa_K2686002) showing bands for the encapsulin multimer high on the gel lanes as well as the monomer around 31kDa, (R) Encapsulin (BBa_K2686001) without HexaHistidine linker, (N) Negative control (cell-free TX-TL expression without DNA and 70C denaturation).

Assembly

The self assembly of the encapsulin 60-mer was first examined using SDS PAGE, where a high band is expected to form due to the high molecular weight and size of the 1.3MDa complex.

SDS PAGE of the different encapsulin proteins expressed by iGEM EPFL 2018. Before (B) heat purification, the pellet after heat purification (P) and the supernatant after heat purification (S). From left to right: 1-3 Negative control (cell-free PURE expression without DNA), 4-6 HexaHis Encapsulin (BBa_K2686002) showing bands for the encapsulin multimer high on the gel lanes as well as the monomer around 31kDa, 7 HexaHis-OVA Encapsulin (BBa_K2686000) where bands are not easily discernible, 8 Ladder, 9 HexaHis-OVA Encapsulin (BBa_K2686000) where the monomer band is visible at 31kDa, 10-12 HexaHis Encapsulin (BBa_K2686002) where the bands for the 60-mer and monomer can be identified, 13-15 HexaHis-OVA Encapsulin (BBa_K2686000) where the bands can easily be discerned for both the monomer and 60-mer (note how the 60-mer band is more visible in the supernatant after heat purification)
SDS PAGE of encapsulins expressed in cell free PURE system with Lysine-BODIPY fluorescent tRNA's. The two different sets of lanes correspond to different heat denaturation temperatures (70C and 100C for 15 minutes). From left to right: N Negative control (cell-free PURE expression without DNA and no purification), L Positive control with DNA coding for Luciferase (37kDa), H HexaHis Encapsulin (BBa_K2686002) showing bands for the encapsulin multimer high on the gel lanes as well as the monomer around 31kDa, R Encapsulin (BBa_K2686001) without HexaHis linker, N Negative control (cell-free PURE expression without DNA and 100C denaturation), Ladder, L Positive control with DNA coding for Luciferase (37kDa), H HexaHis Encapsulin (BBa_K2686002) showing bands for the encapsulin multimer high on the gel lanes as well as the monomer around 31kDa, R Encapsulin (BBa_K2686001) without HexaHis linker, N Negative control (cell-free PURE expression without DNA and 70C denaturation)

References

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.

Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K. and Ueda, T. (2001). Cell-free translation reconstituted with purified components. Nature Biotechnology, 19(8), pp.751-755.

Sun, Z., Hayes, C., Shin, J., Caschera, F., Murray, R. and Noireaux, V. (2013). Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology. Journal of Visualized Experiments, (79).