Part:BBa_K3064014

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. 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.

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===