Difference between revisions of "Part:BBa K3064024"
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===Special Design===
 
===Special Design===
 
Special designs were taken to optimize the applicability and adaptive of such parts.We disassembled the sequence of IgG and fused it with glucagon to get the recombinant glucagon-IgG-Fc.HA Tag was added to N-terminal of Trim21.The two functional modules of the part (glucagon-IgG-Fc and Trim 21) were connected by the linker GGGGS,one of the best fold breaking linkers to expose fusion protein partner well.
 
Special designs were taken to optimize the applicability and adaptive of such parts.We disassembled the sequence of IgG and fused it with glucagon to get the recombinant glucagon-IgG-Fc.HA Tag was added to N-terminal of Trim21.The two functional modules of the part (glucagon-IgG-Fc and Trim 21) were connected by the linker GGGGS,one of the best fold breaking linkers to expose fusion protein partner well.
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https://static.igem.org/mediawiki/parts/6/61/BBa_K3064024.png
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[[File:BBa_K3064024.png|500px|]]
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<B/r>Figure 1.Representation of the function of the composite part.
 
<B/r>Figure 1.Representation of the function of the composite part.
 
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For functional validation, we employed Western Blot, PAS dye, glucose Assay Kit and qPCR. The results shown below illustrate the effectiveness of the part in degrading GCGR, inhibiting glycogenolysis and gluconeogenesis, and decreasing glucose concentration.
 
For functional validation, we employed Western Blot, PAS dye, glucose Assay Kit and qPCR. The results shown below illustrate the effectiveness of the part in degrading GCGR, inhibiting glycogenolysis and gluconeogenesis, and decreasing glucose concentration.
  
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Figure 1. Functional evaluation of the GCGR degradation system in mouse primary hepatocytes after 24h of adenovirus infection.  
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[[File:MmuGlucagon-GGGGS-mmulgG-Fc-P2A-HA-mmuTrim21.jpg|400px|]]
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Figure 2. Functional evaluation of the GCGR degradation system in mouse primary hepatocytes after 24h of adenovirus infection.  
 
(A) Western Blot showing the result of glucagon receptor(GCGR) degradation. (B) PAS dye showing glycogenolysis variation. (C) Cell culture medium glucose concentration measured by glucose Assay Kit. (D) qPCR of crucial enzyme gene PEPCK and G6Pase in gluconeogenesis.
 
(A) Western Blot showing the result of glucagon receptor(GCGR) degradation. (B) PAS dye showing glycogenolysis variation. (C) Cell culture medium glucose concentration measured by glucose Assay Kit. (D) qPCR of crucial enzyme gene PEPCK and G6Pase in gluconeogenesis.
  

Revision as of 15:31, 15 October 2019


mmuGlucagon-GGGGS-mmulgG-Fc-P2A-HA-mmuTrim21

This part is an effective tool for the degradation of glucagon receptor (GCGR).

Usage and Biology

Glucagon is a peptide hormone that generally elevates the concentration of glucose in the blood by promoting gluconeogenesis and glycogenolysis.[1] Immunoglobulin G (IgG) is a type of antibody, of which fragment crystallizable region (Fc) is the tail region. Trim21, an E3 ubiquitin-protein ligase functioning in the intracellular antibody-mediated proteolysis pathway,binds with high affinity to the Fc domain of antibody. HA Tag labels Trim21, making it easier to be detected by Western blotting.P2A is a member of 2A peptides family that can be used to cleave a longer peptide into two shorter peptides after the translation.

With this composite part,the P2A mediated bicistronic expression of the recombinant glucagon-IgG-Fc and Trim 21 can be realized in cells. Glucagon binds to GCGR, Fc domain recruits the Trim21, leading to the ubiquitination and final degradation of GCGR.

Special Design

Special designs were taken to optimize the applicability and adaptive of such parts.We disassembled the sequence of IgG and fused it with glucagon to get the recombinant glucagon-IgG-Fc.HA Tag was added to N-terminal of Trim21.The two functional modules of the part (glucagon-IgG-Fc and Trim 21) were connected by the linker GGGGS,one of the best fold breaking linkers to expose fusion protein partner well. BBa K3064024.png

Figure 1.Representation of the function of the composite part.

Sequence and Features

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 800
    Illegal BamHI site found at 1503
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 901
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 125

Experimental Validation

This part is validated for functional testing.

For functional validation, we employed Western Blot, PAS dye, glucose Assay Kit and qPCR. The results shown below illustrate the effectiveness of the part in degrading GCGR, inhibiting glycogenolysis and gluconeogenesis, and decreasing glucose concentration.


MmuGlucagon-GGGGS-mmulgG-Fc-P2A-HA-mmuTrim21.jpg

Figure 2. Functional evaluation of the GCGR degradation system in mouse primary hepatocytes after 24h of adenovirus infection. (A) Western Blot showing the result of glucagon receptor(GCGR) degradation. (B) PAS dye showing glycogenolysis variation. (C) Cell culture medium glucose concentration measured by glucose Assay Kit. (D) qPCR of crucial enzyme gene PEPCK and G6Pase in gluconeogenesis.



References

1. Voet D, Voet JG (2011). Biochemistry (4th ed.). New York: Wiley.