Difference between revisions of "Part:BBa K2686000"

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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]].  
  
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[[File:LysBODIPYinv.png|thumb|center|upright=4|SDS PAGE of encapsulins expressed in cell free TX-TL system with Lysine-BODIPY fluorescent tRNA's (Fluorotect <nowiki>503nm </nowiki> ). The two different sets of lanes correspond to different heat denaturation temperatures (70C and 100C for 15 minutes). '''Stars''' (<nowiki>&#10022;</nowiki>) show lanes where 60-mers are present.
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[[File:LysBODIPYinv.png|thumb|center|upright=4|SDS PAGE of encapsulins expressed in cell free TX-TL system with Lysine-BODIPY fluorescent tRNA's (Imager settings used were for measuring fluorescein <nowiki>&lambda;<sub>ex</sub> = 494nm, &lambda<sub>em</sub> = 512nm</nowiki> ). The two different sets of lanes correspond to different heat denaturation temperatures (70C and 100C for 15 minutes). '''Stars''' (<nowiki>&#10022;</nowiki>) show lanes where 60-mers are present.
 
From left to right: '''(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 (<nowiki>&#10022;</nowiki>) as well as the monomer around 31kDa, '''(R)''' Encapsulin ([[parts.igem.org/Part:BBa_K2686001|BBa_K2686001]]) without HexaHistidine linker also has a band for the 60-mer (<nowiki>&#10022;</nowiki>), '''(N)''' Negative control (cell-free TX-TL 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 (<nowiki>&#10022;</nowiki>) as well as the monomer around 31kDa, '''(R)''' Encapsulin ([[parts.igem.org/Part:BBa_K2686001|BBa_K2686001]]) without HexaHistidine linker also showing the multimer (<nowiki>&#10022;</nowiki>), '''(N)''' Negative control (cell-free TX-TL expression without DNA and 70C denaturation)]]
 
From left to right: '''(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 (<nowiki>&#10022;</nowiki>) as well as the monomer around 31kDa, '''(R)''' Encapsulin ([[parts.igem.org/Part:BBa_K2686001|BBa_K2686001]]) without HexaHistidine linker also has a band for the 60-mer (<nowiki>&#10022;</nowiki>), '''(N)''' Negative control (cell-free TX-TL 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 (<nowiki>&#10022;</nowiki>) as well as the monomer around 31kDa, '''(R)''' Encapsulin ([[parts.igem.org/Part:BBa_K2686001|BBa_K2686001]]) without HexaHistidine linker also showing the multimer (<nowiki>&#10022;</nowiki>), '''(N)''' Negative control (cell-free TX-TL expression without DNA and 70C denaturation)]]
  

Revision as of 15:54, 16 October 2018


Encapsulin with HexaHistidine insert and C-terminal OT1

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). The C-terminus of the encapsulin is fused to a SIINFEKL (OT1) peptide which is displayed on the exterior surface of the encapsulin monomer as an antigen (Choi et al., 2016). SIINFEKL was chosen as it is a very popular model antigen sequence in research and a variety of antibodies targeting it are available, furthermore it is used as a tumor antigen model in scientific research and has been used in conjunction with encapsulin (Choi et al., 2016).

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

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 (Imager settings used were for measuring fluorescein λ<sub>ex</sub> = 494nm, &lambda<sub>em</sub> = 512nm ). The two different sets of lanes correspond to different heat denaturation temperatures (70C and 100C for 15 minutes). Stars (✦) show lanes where 60-mers are present. From left to right: (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 also has a band for the 60-mer (✦), (N) Negative control (cell-free TX-TL 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 also showing the multimer (✦), (N) Negative control (cell-free TX-TL expression without DNA and 70C denaturation)
SDS PAGE of the different encapsulin proteins expressed by iGEM EPFL 2018. Cell-free mixture Before (B) heat purification procedure outlined above, the pellet after the heat purification procedure (P) and the supernatant after the heat purification procedure (S). Triangles ▲ signify the location of encapsulin 60-mer bands to the right, whereas stars ✦ show encapsulin monomer bands to their right. From left to right: (1-3) Negative control (cell-free TX-TL expression without DNA), (4-6) HexaHistidine Encapsulin (BBa_K2686002) showing bands for the encapsulin multimer high on the gel lanes as well as the monomer around 31kDa, (7) HexaHistidine-OT1 Encapsulin (BBa_K2686000) where bands are not easily discernible, (8) Ladder Precision Plus Protein™ All Blue Prestained Protein Standards #1610373 , (9) HexaHistidine-OT1 Encapsulin (BBa_K2686000) where the monomer band is visible at 31kDa, (10-12) HexaHistidine Encapsulin (BBa_K2686002) where the bands for the 60-mer and monomer can be identified, (13-15) HexaHistidine-OT1 Encapsulin (BBa_K2686000)

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).

Assembly

The self assembly of the encapsulin 60-mer was first examined using PAGE, where the monomer is seen around 32.9kDa as well as a high band due to the high molecular weight and size of the 1.98MDa complex, showing that the 60-mer is present.

Native PAGE gel of sfGFP, HexaHistidine-OT1 Encapsulin and HexaHistidine-OT1 Encapsulin sfGFP-tag. On the left is the fluorescent image of the gel and o the right is the gel after Coomassie staining. Before (B) heat purification procedure outlined in Purification section, Supernatant (S) after the heat purification procedure. Stars ★ show the bands for monomers of HexaHistidine-OT1 Encapsulin (BBa_K2686000,BBa_K2686006) with sfGFP-tag bound to them, increasing the molecular weight of the complex when compared to sfGFP alone. Triangles ▲ signify a 60-mer band to the right. From left to right: (1-2) Negative control (cell-free TX-TL expression without DNA), (2-4) HexaHistidine-OT1 Encapsulin (BBa_K2686000), showing no fluorescence on the left gel, has a band for 60-mer on stained gel (▲). (5-6) sfGFP protein, the fluorescent bands can be easily seen as large smears on the left. (7-8) HexaHistidine-OT1 Encapsulin and sfGFP-tag (BBa_K2686006) shown by a ★. The difference in height between the bands of sfGFP compared to the ★ is striking and suggests that the sfGFP-tag binds to the HexaHistidine-OT1 Encapsulin monomers. In addition there seems to be a small amount of 60-mer indicated by ▲. (9-10) Encapsulin (BBa_K2686001) does assemble to form the 60-mer ▲ as seen on the stained gel. (11) HexaHistidine-OT1 Encapsulin and sfGFP-tag (BBa_K2686006), on the left the presumed ★ protein dimer is seen to be higher than the sfGFP in lane 12, no particular bands can be identified on the right. (12) sfGFP, fluorescent band is seen to be lower than for lane 11.

References

Choi, B., Moon, H., Hong, S., Shin, C., Do, Y., Ryu, S. and Kang, S. (2016). Effective Delivery of Antigen–Encapsulin Nanoparticle Fusions to Dendritic Cells Leads to Antigen-Specific Cytotoxic T Cell Activation and Tumor Rejection. ACS Nano, 10(8), pp.7339-7350.

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).