Difference between revisions of "Part:BBa K3064014"
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<partinfo>BBa_K3064000 short</partinfo>
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<partinfo>BBa_K3064014 short</partinfo>
  
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It is a binding site of CHREBP.
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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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Some glycolytic and lipogenic genes respond primarily to glucose, not to insulin. This led to the identification of a consensus sequence that is required for glucose-responsiveness, termed the carbohydrate response element (ChoRE), comprising of two E-boxes (CACGTG) or E-box-like sequences separated by 5 bp [3-5].
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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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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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Carbohydrate response element binding protein (ChREBP) is an important transcription that can regulate the expression of key genes involved in various pathways including glycolysis, gluconeogenesis and lipogenesis. It does this by forming a tetrameric complex made up of two ChREBP/Mlx heterodimers, which enables it to bind to the carbohydrate response element (ChoRE) in the promoter region. ChoRE should be widely applicable to studying the influence of glucose consentration to cells .
 
  
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https://static.igem.org/mediawiki/parts/4/4c/T--NUDT_CHINA--part-CHoRE_1.jpg
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===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 . 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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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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Figure 1: ChREBP bind with ChoRE
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[[File:T--NUDT_CHINA--hsaGlucagon-design_.jpg|500px]]
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The basic part BBa_K3064000 is an important part in our next experiment. The different number of CHoRE are set in other parts. And the part BBa_K3064026 shows his function. This part BBa_K3064026 was cloned in pGL3 and transfected into HepG2 cell lines using Invitrogen LipofectamineTM 3000. Protocol could be found in our Experiment page.
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<p><ahref="https://2019.igem.org/wiki/images/1/1b/T--NUDT_CHINA--Protocol_for_lipo3000_transfection_with_Lipofectamine%E2%84%A2_3000_Reagent.pdf">Click to see </ a></p >
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To conduct the later function test, we set five different groups to conduct the transfection. They are pGL3-9xGSP(glucogon sensor promoter)-GFP , pGL3-6xGSP-GFP, pGL3-3xGSP-GFP, pGL3-minip-GFPand plv-mcherry as the internal control. We transfected 300μg into HepG2 cells, which were cultured on 24-hole plate. When 90 percent  were mixed, we begin the transfection.
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At the very first beginning, we starved the HepG2 cells with DMEM for 2 hours before transfection begins.After transfection 12h, we starved the cells with glucose-free culture and stimulate with 20mM glucose concentration culture after 6 more hours. Glucose stimulation intensity was controlled at 20mM. Samples were tested after transfection of 48h.
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Figure 1.Representation of the function of the composite part.
  
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===Sequence and Features===
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3064000 SequenceAndFeatures</partinfo>
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<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_K3064000 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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==Results==
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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.
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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.
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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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===Contribution:NUDT_CHINA, 2020===
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===Method===
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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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===Functional Test===
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===Result===
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After 18 hours’ transfection, we conduct experiments to test the function of our part. Photograph of fluorescence microscopy helps make results clear and obvious. Meanwhile, with the set of internal control, we can gain relative fluorescence intensity by Image J.  During this process. First, we set different groups with different promoter so that we can understand that the number of CHoRE can influence the promoter's efficiency. And we know that the more CHoRE has, the better effect we have.
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Then, we set the experiment that 6xGsp-GFP in different glucose concentration for 48 hours'. And we get the result that the effect is different in different glucose concentration.
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https://static.igem.org/mediawiki/parts/c/c1/T--NUDT_CHINA--CHoRE-result.png
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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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Figure 2: Some structures and results on CHoRE
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<html>
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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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Figure 2 (A): Some strutures on CHoRE. Figure 2 (B): minip/3xGsp/6xGsp/9xGsp after 48 hours' transfection in 10 um glucose stimulation. Figure 2 (C): 6xGsp in different glucose concentration after 48 hours.
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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).