Difference between revisions of "Part:BBa K3064014"
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===Functional Test===
 
===Functional Test===
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This composite part was constructed with its basic coding part (BBa_K1997005), lac promoter+RBS (BBa_K1789012), and double terminator (BBa_B0015). Thus, the function of this composite part was validated together with BBa_K1997005.
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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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The basic function of our split-HRP reporting system has been determined in the function validation of BBa_K1997013 and BBa_K1997012. Building on that, BBa_K1997011 was built to demonstrate the imbedded substitution system. For such matters, the Zif268 region in BBa_K1997012 was replaced into a “FRB-RBS-FKBP” fragment using Golden Gate Assembly. The cloning results were validated through sequencing.
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For functional validation, we used Rapamycin to induce the interaction between FRB and FKBP. For such assay, E.coli carrying respective plasmid was cultured overnight under IPTG induction. Cells were then collected and lysed by high-pressure homogenizer. Once lysed and ultra-filtrated (to remove small molecules), 0.4nM of Rapamycin was added together with TMB substrate solution (with heme supplementation) into the cell lysate for the induction of protein-protein interaction and measurement of HRP activity. The plate was then incubated under 37°C for 5min before adding the Stop solution.
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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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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.  
  
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Value of OD<sub>450 </sub> obtained after adding the Stop Solution showed significant variation between the Rapamycin positive group and the Rapamycin negative group. Thus then validated the function of this part.
 
 
https://static.igem.org/mediawiki/parts/c/c0/T--NUDT_CHINA--BBa_K3064014_.jpg
 
https://static.igem.org/mediawiki/parts/c/c0/T--NUDT_CHINA--BBa_K3064014_.jpg
  
 
Figure 2. Functional evaluation of the GCGR degradation system in mouse primary hepatocytes HepG2 cells after 12h of liposome transfection.  
 
Figure 2. Functional evaluation of the GCGR degradation system in mouse primary hepatocytes 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.(C) Cell culture medium glucose concentration measured by glucose Assay Kit. (D) PAS dye showing glycogenolysis variation. (E) RNA abundance test showing GCGR mRNA variation. (F) qPCR of crucial enzyme gene PEPCK and G6Pase in gluconeogenesis.
 
(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.(C) Cell culture medium glucose concentration measured by glucose Assay Kit. (D) PAS dye showing glycogenolysis variation. (E) RNA abundance test showing GCGR mRNA variation. (F) qPCR of crucial enzyme gene PEPCK and G6Pase in gluconeogenesis.
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===References===

Revision as of 02:45, 16 October 2019


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

This is a functional unit of the HepG2, which expresses fusioned hsaGlucagon-GGGGS-hsaIgG-Fc and HA-hsaTrim21 by automatic cleavage of P2A.

Usage and Biology

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.
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.
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.

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 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. T--NUDT_CHINA--BBa_K3064014-design1_.jpg T--NUDT_CHINA--BBa_K3064014-design2_.jpg Figure 1.Representation of the function of the composite part.

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


Functional Test

This composite part is to achieve ubiquitination of GCGR. To verify whether it worked , we did a functional test of it.

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.

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.

T--NUDT_CHINA--BBa_K3064014_.jpg

Figure 2. Functional evaluation of the GCGR degradation system in mouse primary hepatocytes 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.(C) Cell culture medium glucose concentration measured by glucose Assay Kit. (D) PAS dye showing glycogenolysis variation. (E) RNA abundance test showing GCGR mRNA variation. (F) qPCR of crucial enzyme gene PEPCK and G6Pase in gluconeogenesis.

===References===