Difference between revisions of "Part:BBa K3064014"
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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. | 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. | 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. | 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. | ||
Revision as of 03:58, 22 October 2019
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.
Figure 1.Representation of the function of the composite part.
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1830
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2499
Illegal BamHI site found at 3037 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1787
Illegal AgeI site found at 632 - 1000INCOMPATIBLE 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.
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.