Difference between revisions of "Part:BBa K2686001"

Revision as of 11:03, 17 October 2018

Encapsulin protein

This is a BioBrick containing the sequence for Thermotoga maritima encapsulin, a bacterial protein nanocompartment which self assembles to form a 60-mer.

Usage and Biology

Encapsulins are versatile proteins found in a variety of different bacteria (Giessen and Silver, 2017). In the case of this specific part derived from Thermotoga maritima, it can be used among other things to deliver cargo, both on the outer surface of the nanoparticle by fusing a peptide in between the 139/140 Amino Acids or the protein's C terminus. A cargo protein can also be loaded by fusing it with a tag binding to Encapsulin's interior surface (Cassidy-Amstutz 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 441
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 375
Illegal SapI.rc site found at 406

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_ex=494nm, λ_em=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)

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 SDS PAGE, where a band around 30.71kDa band is expected to form. Additionally due to Encapsulin's exceptional heat stability the 1.98MDa complex also appears on the gel after SDS denaturation.

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

Cassidy-Amstutz, C., Oltrogge, L., Going, C., Lee, A., Teng, P., Quintanilla, D., East-Seletsky, A., Williams, E. and Savage, D. (2016). Identification of a Minimal Peptide Tag for in Vivo and in Vitro Loading of Encapsulin. Biochemistry, 55(24), pp.3461-3468.

Giessen, T. and Silver, P. (2017). Widespread distribution of encapsulin nanocompartments reveals functional diversity. Nature Microbiology, 2, p.17029.

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.

@@ Line 33: / Line 33: @@
 The self assembly of the encapsulin 60-mer was first examined using SDS PAGE, where a band around 30.71kDa band is expected to form. Additionally due to Encapsulin's exceptional heat stability the 1.98MDa complex also appears on the gel after SDS denaturation.
-[[File:NativeGel.png|thumb|center|upright=3|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''' <nowiki>&#9733;</nowiki> show the bands for monomers of HexaHistidine-OT1 Encapsulin ([[parts.igem.org/Part:BBa_K2686002|BBa_K2686000]],[[parts.igem.org/Part:BBa_K2686006|BBa_K2686006]]) with sfGFP-tag bound to them, increasing the molecular weight of the complex when compared to sfGFP alone. '''Triangles''' <nowiki>&#9650;</nowiki> signify a 60-mer band to the right.
+[[File:NativeGel.png|thumb|center|upright=3|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''' <nowiki>&#9733;</nowiki> show the bands for monomers of HexaHistidine-OT1 Encapsulin (<bbpart>BBa_K2686000</bbpart>,<bbpart>BBa_K2686006</bbpart>) with sfGFP-tag bound to them, increasing the molecular weight of the complex when compared to sfGFP alone. '''Triangles''' <nowiki>&#9650;</nowiki> 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 ([[parts.igem.org/Part:BBa_K2686000|BBa_K2686000]]), showing no fluorescence on the left gel, has a band for 60-mer on stained gel (<nowiki>&#9650;</nowiki>). '''(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 ([[parts.igem.org/Part:BBa_K2686006|BBa_K2686006]]) shown by a <nowiki>&#9733;</nowiki>. The difference in height between the bands of sfGFP compared to the <nowiki>&#9733;</nowiki> 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 <nowiki>&#9650;</nowiki>. '''(9-10)''' Encapsulin ([[parts.igem.org/Part:BBa_K2686001|BBa_K2686001]]) does assemble to form the 60-mer <nowiki>&#9650;</nowiki> as seen on the stained gel. '''(11)''' HexaHistidine-OT1 Encapsulin and sfGFP-tag ([[parts.igem.org/Part:BBa_K2686006|BBa_K2686006]]), on the left the presumed <nowiki>&#9733;</nowiki> 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'''.
+From left to right: '''(1-2)''' Negative control (cell-free TX-TL expression without DNA), '''(2-4)''' HexaHistidine-OT1 Encapsulin (<bbpart>BBa_K2686000</bbpart>), showing no fluorescence on the left gel, has a band for 60-mer on stained gel (<nowiki>&#9650;</nowiki>). '''(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 (<bbpart>BBa_K2686006</bbpart>) shown by a <nowiki>&#9733;</nowiki>. The difference in height between the bands of sfGFP compared to the <nowiki>&#9733;</nowiki> 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 <nowiki>&#9650;</nowiki>. '''(9-10)''' Encapsulin (<bbpart>BBa_K2686001</bbpart>) does assemble to form the 60-mer <nowiki>&#9650;</nowiki> as seen on the stained gel. '''(11)''' HexaHistidine-OT1 Encapsulin and sfGFP-tag (<bbpart>BBa_K2686006</bbpart>), on the left the presumed <nowiki>&#9733;</nowiki> 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'''.
 ]]