Difference between revisions of "Part:BBa K4143337"
 
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<partinfo>BBa_K4143337 short</partinfo>
 
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Encapsulins are protein compartments that exist naturally in a variety of bacteria and archaea, and are composed of a single shell protein that self-assembles and can help to sequester and protect antimicrobial peptides when they are expressed in E.coli. Once assembled, they typically form 25–42 nm diameter icosahedral structures.
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Encapsulins are protein nanocompartments that exist naturally in a variety of bacteria and archaea. They are composed of single shell monomer proteins that self-assemble into icosahedral geometries. They are able to sequester cargo (such as antimicrobial peptides) carrying a targeting peptide (BBa_K4143339 when they are expressed in E.coli. Once assembled, they typically form 25–42 nm diameter icosahedral structures [1]. This part encodes the sequence for a T4GALA encapsulin monomer.  
  
 
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===Usage and Biology===
 
===Usage and Biology===
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Encapsulins are protein organelles that encapsulate cargo proteins which are "targeted to the encapsulin capsid interior via small C-terminal peptides referred to as targeting peptides" [1]. They typically function to sequester harmful reactions from the rest of the cell, enabling otherwise incompatible reactions to occur in a prokaryotic cell at the same time. The encapsulin system consists of the encapsulin capsid and core cargo genes, which may also be referred to as the core operon.
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Encapsulins are notable for being stable once assembled, which prevents the efficient release of cargo, therefore physiological conditions such as surrounding pH conditions may prompt disassembly. Notably, "disassembling and reassembling viral capsids or encapsulins requires extremes of pH or salt concentration” [2].
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The T4GALA encapsulin is unique in that it can be disassembled by simply lowering the environmental pH to 6. This facilitates convenient release of cargo from the encapsulin. This encapsulin is derived from Q. thermotolerans and consists of 240 subunits. We intend to use this part along with our targeting peptide (BBa_K4143339), antimicrobial peptide (AMP) HBCM2 (BBa_K4143336) to facilitate the expression of an otherwise toxic peptide. Composite part BBa_K4143340 contains the details for this system.
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<h4>PCR Amplification</h4>
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For our project, we aimed to use encapsulins to facilitate the expression of toxic peptides such as antimicrobial peptides. To do so, we first amplified the encapsulin from a vector containing the encapsulin monomer gene. To confirm PCR was successful, we ran agarose gel electrophoresis and confirmed our PCR product matched the expected size (994 bases).
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[[File:enc-gel.png|300px|thumb|left|Figure 1: Agarose gel electrophoresis of T4GALA encapsulin shown in lane 5. ]]
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<h4>Protein Purification</h4>
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After expressing the encapsulin in BL21 E. coli, we purified the expression product and confirmed the encapsulin’s presence using an SDS-PAGE gel. We knew the size of the encapsulin was approximately 32 kDa, and this matched the band on the gel at this size on the ladder.
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[[File:results-5-updated.png|550px|thumb|left|Figure 2: SDS-PAGE gel of AMP (HBCM2) + encapsulin. Purified encapsulin is shown at approximately the middle of the gel around the 32 kDa mark.]]
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===References===
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1. T. W. Giessen and P. A. Silver, “Widespread distribution of encapsulin nanocompartments reveals functional diversity,” Nature Microbiology, vol. 2, no. 6, p. 17029, Mar. 2017, doi: 10.1038/nmicrobiol.2017.29.
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2. J. A. Jones, A. S. Cristie-David, M. P. Andreas, and T. W. Giessen, “Triggered reversible disassembly of an engineered protein nanocage,” bioRxiv, p. 2021.04.19.440480, Jan. 2021, doi: 10.1101/2021.04.19.440480.
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Encapsulins are protein organelles, that encapsulate cargo proteins which are "targeted to the encapsulin capsid interior via small C-terminal peptides referred to as targeting peptides." (Giessen) They typically function to sequester harmful reactions from the rest of the cell, which means that incompatible reactions can occur in a cell at the same time. The encapsulin system is consists of the encapsulin capsid and core cargo genes, which may also be referred to as the core operon.
 
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Latest revision as of 15:04, 11 October 2022


T4GALA Encapsulin

Encapsulins are protein nanocompartments that exist naturally in a variety of bacteria and archaea. They are composed of single shell monomer proteins that self-assemble into icosahedral geometries. They are able to sequester cargo (such as antimicrobial peptides) carrying a targeting peptide (BBa_K4143339 when they are expressed in E.coli. Once assembled, they typically form 25–42 nm diameter icosahedral structures [1]. This part encodes the sequence for a T4GALA encapsulin monomer.

Usage and Biology

Encapsulins are protein organelles that encapsulate cargo proteins which are "targeted to the encapsulin capsid interior via small C-terminal peptides referred to as targeting peptides" [1]. They typically function to sequester harmful reactions from the rest of the cell, enabling otherwise incompatible reactions to occur in a prokaryotic cell at the same time. The encapsulin system consists of the encapsulin capsid and core cargo genes, which may also be referred to as the core operon.

Encapsulins are notable for being stable once assembled, which prevents the efficient release of cargo, therefore physiological conditions such as surrounding pH conditions may prompt disassembly. Notably, "disassembling and reassembling viral capsids or encapsulins requires extremes of pH or salt concentration” [2].

The T4GALA encapsulin is unique in that it can be disassembled by simply lowering the environmental pH to 6. This facilitates convenient release of cargo from the encapsulin. This encapsulin is derived from Q. thermotolerans and consists of 240 subunits. We intend to use this part along with our targeting peptide (BBa_K4143339), antimicrobial peptide (AMP) HBCM2 (BBa_K4143336) to facilitate the expression of an otherwise toxic peptide. Composite part BBa_K4143340 contains the details for this system.

PCR Amplification

For our project, we aimed to use encapsulins to facilitate the expression of toxic peptides such as antimicrobial peptides. To do so, we first amplified the encapsulin from a vector containing the encapsulin monomer gene. To confirm PCR was successful, we ran agarose gel electrophoresis and confirmed our PCR product matched the expected size (994 bases).

Figure 1: Agarose gel electrophoresis of T4GALA encapsulin shown in lane 5.


Protein Purification

After expressing the encapsulin in BL21 E. coli, we purified the expression product and confirmed the encapsulin’s presence using an SDS-PAGE gel. We knew the size of the encapsulin was approximately 32 kDa, and this matched the band on the gel at this size on the ladder.

Figure 2: SDS-PAGE gel of AMP (HBCM2) + encapsulin. Purified encapsulin is shown at approximately the middle of the gel around the 32 kDa mark.


References

1. T. W. Giessen and P. A. Silver, “Widespread distribution of encapsulin nanocompartments reveals functional diversity,” Nature Microbiology, vol. 2, no. 6, p. 17029, Mar. 2017, doi: 10.1038/nmicrobiol.2017.29.

2. J. A. Jones, A. S. Cristie-David, M. P. Andreas, and T. W. Giessen, “Triggered reversible disassembly of an engineered protein nanocage,” bioRxiv, p. 2021.04.19.440480, Jan. 2021, doi: 10.1101/2021.04.19.440480.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]