Difference between revisions of "Part:BBa K3064014"

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<partinfo>BBa_K3064014 short</partinfo>
 
<partinfo>BBa_K3064014 short</partinfo>
  
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This is a functional unit of the HepG2, which expresses fusioned hsaGlucagon-GGGGS-hsaIgG-Fc and HA-hsaTrim21 by automatic cleavage of P2A.
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This part is used to degrade glucagon receptor (GCGR) endogenously.
  
 
<!-- Add more about the biology of this part here -->
 
<!-- Add more about the biology of this part here -->
 
===Usage and Biology===
 
===Usage and Biology===
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Our composite part BBa_K3064014 play a necessary effect in our whole RECEPTOR KILLER system. This part is designed to recognised and bind with target protein,and then recruit the proteasome to deplete the target protein through the ubiquitin-proteasome pathway.<Br/>
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In this part, hsaGlucagon acts as a bait,which can bind with GCGR, and is linked together with hsalgG-Fc by the designed 4xGS linker to form a recombinate GFP antibody.<Br/>
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Our composite part BBa_K3064014 play a necessary effect in our whole GCGR Predator system. This part is designed to recognised and bind with GCGR protein,and then recruit the proteasome to deplete the target protein through the ubiquitin-proteasome pathway.
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Trim21, the E3 ubiquitin ligase, plays the most important role in the degradation system. The C-terminal B30.2 domain on trim21 offers a site for the conservative Fc region of human lgG 1,2 and 4 to bind with[1]. When Glucagon is linked to the hsalgG1-Fc, and after the antibody-antigen interaction, a ternary complex is build up. The trim21 then functions as a E3 ubiquitin ligase and proceeds the complex to be depleted through the ubiquitin-proteasome pathway.  
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In this part, hsaGlucagon interacts with GCGR in a ligand-receptor binding manner. Trim21, the E3 ubiquitin ligase, plays the most important role in the degradation system. The C-terminal B30.2 domain on trim21 offers a site for the conservative Fc region of human lgG[1]. The trim21 then functions as a E3 ubiquitin ligase and proceeds the complex to be depleted through the ubiquitin-proteasome pathway.
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===Special Design===
 
===Special Design===
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As a key functional element, special designs are taken for to optimize the applicability and adaptive of such parts
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As a key functional element, special designs are taken for to optimize the applicability and adaptive of such parts . Our part is mainly composed of two important genes, and the efficient expression of these two genes and how to connect them become the focus of our design.
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. Our part is mainly composed of two important genes, and the efficient expression of these two genes and how to connect them become the focus of our design.<Br/>
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Firstly, we used a connection element named GGGGS(BBa K3064025) to connect hsaGlucagon to hsalgG-fc. One of them is the specific binding site of the target receptor--GCGR. The other is the binding part of Trim21(BBa_K2653000). Second, we used self-cleaved sequence named P2A(BBa_K2653003), which allowed us to express two important genes at the same time. Make our parts more efficient. We also integrated HA tag(BBa_K2653004) into the Trim21 gene, which allows easy detection of Trim21 expression by Western Blotting analysis.
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Firstly, we use a connection element named GGGGS(BBa K3064025), which connects hsaGlucagon to hsalgG-fc. One of them is the specific binding site of the target receptor--GCGR. The other is the binding part of Trim21(BBa_K2653000).second, it is a kind of self-cleaved sequence named P2A(BBa_K2653003). It allows our part to express two important genes at the same time. Make our parts more efficient. HA(BBa_K2653004), after it, is a marker protein. It allows us to determine whether the protein translated by part is our target protein, which makes our experiment more accurate.
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https://static.igem.org/mediawiki/parts/e/e8/T--NUDT_CHINA--BBa_K3064014-design1_.jpg
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[[File:T--NUDT_CHINA--hsaGlucagon-design_.jpg|500px]]
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https://static.igem.org/mediawiki/parts/3/3f/T--NUDT_CHINA--BBa_K3064014-design2_.jpg
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<B/r>Figure 1.Representation of the function of the composite part.
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Figure 1.Representation of the function of the composite part.
  
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<!-- -->
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===Sequence and Features===
 
<!-- -->
 
<!-- -->
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3064014 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3064014 SequenceAndFeatures</partinfo>
  
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===Characterization===
  
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<!-- Uncomment this to enable Functional Parameter display
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This composite part can induce glucose concentration and achieve ubiquitination of GCGR. To verify whether it worked, we did a test of it.
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===Functional Parameters===
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<partinfo>BBa_K3064014 parameters</partinfo>
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==Methods==
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<!-- -->
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HepG2 cells were transfected with Gcg-Fc expressing cassette or GCGR Predator (GCGR Pr) expressing cassette. Immunoprecipitation was performed with IgG-Fc targeting Protein A/G beads 48 h posttranscription followed up by Western blotting analysis probing GCGR and the Gcg-Fc domain of the GCGR Predator. Western Blotting is also performed to determine the degradation of GCGR.
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===Functional Test===
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This composite part is to achieve ubiquitination of GCGR. To verify whether it worked , we did a functional test of it.
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 +
==Results==
 +
First, we used the method of immunoprecipitation to determine whether lgG-Fc will influence glucagon's function. And the experimental result shows that glucagon still binds with GCGR very well, which proved the integrity of this functional element.
 +
Then we have a function experiment to compared it to a control group. The results shown below illustrate the effectiveness of the part in degrading GCGR, inhibiting glycogenolysis and gluconeogenesis, and decreasing glucose concentration.
 +
 
 +
https://static.igem.org/mediawiki/parts/3/3a/T--NUDT_CHINA--part-hsa_.jpg
 +
 
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Figure 2. Functional evaluation of the GCGR degradation system in HepG2 cells after 12h of liposome transfection. (A) Co-immunoprecipitation showing binding ability of glucagon-humanIgG Fc fusion protein with glucagon receptor(GCGR). (B) Western Blot showing the result of glucagon receptor(GCGR) degradation.
  
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First, we used the method of immunoprecipitation to determine whether it was properly translated in the primary hepatocyte system as we wanted. The experimental results were good, which proved the integrity of this functional element.
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===Contribution:NUDT_CHINA, 2020===
  
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Then we have an experiment to compared it to a control group. The results shown below illustrate the effectiveness of the part in degrading GCGR, inhibiting glycogenolysis and gluconeogenesis, and decreasing glucose concentration.  
+
===Method===
 +
To further validate that intracellular glucagon can be degraded by GCGR Predator, HepG2 cells were transfected with Trim21 expression cassette, Glucagon-Fc expressing cassette(this part) or GCGR Predator (GCGR Pre.) expressing cassette, and Western blotting analysis was performed to detect the protein level of GCGR(Fig 3A).
  
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https://static.igem.org/mediawiki/parts/1/1f/T--NUDT_CHINA--silver_2_result.jpg
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===Result===
  
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Figure 2. Functional evaluation of the GCGR degradation system in HepG2 cells after 12h of liposome transfection.
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We performed Western Blotting to prove that both Trim21 and Glucagon-linker-IgG Fc (Gcg-Fc) is required to effectively trigger GCGR degradation. Results indicated that GCGR Pre. transfected cells showed a ~40% lower GCGR level compared to the vector control (Fig 3A and B). Besides, there is no significant difference between the control group and groups that only overexpressed Gcg-Fc or Trim21. Also, we found that both in four groups, Glycosylated GCGR (Glu-GCGR, ~64kDa) showed no difference in expression level. These results demonstrated that both Trim21 and Gcg-Fc are needed to achieve effective degradation of target protein GCGR.
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(A) Co-immunoprecipitation showing binding ability of glucagon-humanIgG Fc fusion protein with glucagon receptor(GCGR). (B) Western Blot showing the result of glucagon receptor(GCGR) degradation.
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This part expresses two separate crucial proteins:IgG-Fc fusioned glucagon and HA tagged Trim21. These two parts combines together through binding ability of trim21 N terminal to IgG-Fc. Meanwhile, GCGR binds with glucagon and these three parts form a ternary complex. When trim21 functions as an E3 ubiquitin ligase, the complex would be ubiquitin tagged and degraded through the proteasome pathway. In this way, intracellular GCGR content in hepatocytes would decrease, and so would those distributed in cell membranes.  
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<html>
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The highlight of this part is that the glucagon in this part can be replaced. It could be an antibody, or it could be another ligand, or else. Almost any protein is possible to be directly degraded in cell if the part expresses some fragments that could binds with the target.
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<p><img src="https://2020.igem.org/wiki/images/2/29/T--NUDT_CHINA--ContributionFig2-2.jpg" alt="" width="900"/></p>
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</html>
  
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===References===
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Figure 3. (A) Western blotting assay showing the degradation of GCGR in the GCGR Predator transfected group and its quantified results (B).

Latest revision as of 16:47, 27 October 2020


hsaGlucagon->GGGGS->hsaIgG-Fc->P2A->HA->hsaTrim21

This part is used to degrade glucagon receptor (GCGR) endogenously.

Usage and Biology

Our composite part BBa_K3064014 play a necessary effect in our whole GCGR Predator system. This part is designed to recognised and bind with GCGR protein,and then recruit the proteasome to deplete the target protein through the ubiquitin-proteasome pathway. In this part, hsaGlucagon interacts with GCGR in a ligand-receptor binding manner. Trim21, the E3 ubiquitin ligase, plays the most important role in the degradation system. The C-terminal B30.2 domain on trim21 offers a site for the conservative Fc region of human lgG[1]. The trim21 then functions as a E3 ubiquitin ligase and proceeds the complex to be depleted through the ubiquitin-proteasome pathway.


Special Design

As a key functional element, special designs are taken for to optimize the applicability and adaptive of such parts . Our part is mainly composed of two important genes, and the efficient expression of these two genes and how to connect them become the focus of our design. Firstly, we used a connection element named GGGGS(BBa K3064025) to connect hsaGlucagon to hsalgG-fc. One of them is the specific binding site of the target receptor--GCGR. The other is the binding part of Trim21(BBa_K2653000). Second, we used self-cleaved sequence named P2A(BBa_K2653003), which allowed us to express two important genes at the same time. Make our parts more efficient. We also integrated HA tag(BBa_K2653004) into the Trim21 gene, which allows easy detection of Trim21 expression by Western Blotting analysis.

T--NUDT CHINA--hsaGlucagon-design .jpg


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


Sequence and Features

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1830
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 2499
    Illegal BamHI site found at 3037
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1787
    Illegal AgeI site found at 632
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1142
    Illegal BsaI site found at 2877
    Illegal SapI.rc site found at 1490

Characterization

This composite part can induce glucose concentration and achieve ubiquitination of GCGR. To verify whether it worked, we did a test of it.

Methods

HepG2 cells were transfected with Gcg-Fc expressing cassette or GCGR Predator (GCGR Pr) expressing cassette. Immunoprecipitation was performed with IgG-Fc targeting Protein A/G beads 48 h posttranscription followed up by Western blotting analysis probing GCGR and the Gcg-Fc domain of the GCGR Predator. Western Blotting is also performed to determine the degradation of GCGR.


Results

First, we used the method of immunoprecipitation to determine whether lgG-Fc will influence glucagon's function. And the experimental result shows that glucagon still binds with GCGR very well, which proved the integrity of this functional element. Then we have a function experiment to compared it to a control group. The results shown below illustrate the effectiveness of the part in degrading GCGR, inhibiting glycogenolysis and gluconeogenesis, and decreasing glucose concentration.

T--NUDT_CHINA--part-hsa_.jpg

Figure 2. Functional evaluation of the GCGR degradation system in HepG2 cells after 12h of liposome transfection. (A) Co-immunoprecipitation showing binding ability of glucagon-humanIgG Fc fusion protein with glucagon receptor(GCGR). (B) Western Blot showing the result of glucagon receptor(GCGR) degradation.

Contribution:NUDT_CHINA, 2020

Method

To further validate that intracellular glucagon can be degraded by GCGR Predator, HepG2 cells were transfected with Trim21 expression cassette, Glucagon-Fc expressing cassette(this part) or GCGR Predator (GCGR Pre.) expressing cassette, and Western blotting analysis was performed to detect the protein level of GCGR(Fig 3A).

Result

We performed Western Blotting to prove that both Trim21 and Glucagon-linker-IgG Fc (Gcg-Fc) is required to effectively trigger GCGR degradation. Results indicated that GCGR Pre. transfected cells showed a ~40% lower GCGR level compared to the vector control (Fig 3A and B). Besides, there is no significant difference between the control group and groups that only overexpressed Gcg-Fc or Trim21. Also, we found that both in four groups, Glycosylated GCGR (Glu-GCGR, ~64kDa) showed no difference in expression level. These results demonstrated that both Trim21 and Gcg-Fc are needed to achieve effective degradation of target protein GCGR.

Figure 3. (A) Western blotting assay showing the degradation of GCGR in the GCGR Predator transfected group and its quantified results (B).