Difference between revisions of "Part:BBa K3064024"

Revision as of 10:59, 15 October 2019

mmuGlucagon-GGGGS-mmulgG-Fc-P2A-HA-mmuTrim21

This part is an effective tool for the degradation of glucagon receptor (GCGR).

Usage and Biology

Glucagon is a peptide hormone that generally elevates the concentration of glucose in the blood by promoting gluconeogenesis and glycogenolysis.[1] Immunoglobulin G (IgG) is a type of antibody, of which fragment crystallizable region (Fc) is the tail region. Trim21, an E3 ubiquitin-protein ligase functioning in the intracellular antibody-mediated proteolysis pathway,binds with high affinity to the Fc domain of antibody. HA Tag labels Trim21, making it easier to be detected by Western blotting.P2A is a member of 2A peptides family that can be used to cleave a longer peptide into two shorter peptides after the translation.

With this composite part,the P2A mediated bicistronic expression of the recombinant glucagon-IgG-Fc and Trim 21 can be realized in cells. Glucagon binds to GCGR, Fc domain recruits the Trim21, leading to the ubiquitination and final degradation of GCGR.

Special Design

Special designs were taken to optimize the applicability and adaptive of such parts.We disassembled the sequence of IgG and fused it with glucagon to get the recombinant glucagon-IgG-Fc.HA Tag was added to N-terminal of Trim21.The two functional modules of the part (glucagon-IgG-Fc and Trim 21) were connected by the linker GGGGS,one of the best fold breaking linkers to expose fusion protein partner well. Figure 1.Representation of the function of the composite part. Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]

12
COMPATIBLE WITH RFC[12]

21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 800
Illegal BamHI site found at 1503

23
COMPATIBLE WITH RFC[23]

25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 901

1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 125

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 <partinfo>BBa_K3064024 short</partinfo>
-This composite part is made up of six basic parts:mmuGlucagon,GGGGS,mmuIgG-Fc,P2A,HA and mmuTrim21. <Br/>
+This part is an effective tool for the degradation of glucagon receptor (GCGR).
-It includes the parts we need to construct a GCGR degradation system. mmuGlucagon can bing with glucagon receptor specifically and it plays a role as a navigator in the composite part. GGGGS Linker is mostly used as a linker for protein and it is one of the best fold breaking linker to expose fusion protein partner well.Immunoglobulin G (IgG) is a type of antibody, which binds many kinds of pathogens and protects the body from infection. Also, it can be recognized and bound with TRIM21,an E3 ubiquitin-protein ligase that has been applied to a protein degradation method known as Trim-Away.The bicistronic P2A peptide is a kind of self-cleaved sequence, which breaks when translated. It can be used to construct the polycis vector and realize the co-translation of two proteins under the control of the same promoter.The HA-tag we use in our system has the length of 27 bps. It is a really useful tool to link antibody and Trim21.Trim21, as referred,can trigger the degradation of certain protein.<Br/>
-Composed of the basic parts above, this composite part can degrade GCGR specifically and thus lower glucose content to realize our goal.
 <!-- Add more about the biology of this part here-->
 ===Usage and Biology===
-We use this composite part to degrade the content of glucagon-receptor specifically which can lead to the decrease of glucose content in mice and cause the change of hepatic metabolism. By observing the changes we can further explore the relationship between two symptoms.
+Glucagon is a peptide hormone that generally elevates the concentration of glucose in the blood by promoting gluconeogenesis and glycogenolysis.[1] Immunoglobulin G (IgG) is a type of antibody, of which fragment crystallizable region (Fc) is the tail region.
+Trim21, an E3 ubiquitin-protein ligase functioning in the intracellular antibody-mediated proteolysis pathway,binds with high affinity to the Fc domain of antibody. HA Tag labels Trim21, making it easier to be detected by Western blotting.P2A is a member of 2A peptides family that can be used to cleave a longer peptide into two shorter peptides after the translation.
+With this composite part,the P2A mediated bicistronic expression of the recombinant glucagon-IgG-Fc and Trim 21 can be realized in cells. Glucagon binds to GCGR, Fc domain recruits the Trim21, leading to the ubiquitination and final degradation of GCGR.
 ===Special Design===
-Special designs were taken to optimize the applicability and adaptive of such parts. GGGGS is one of the best fold breaking linker to expose fusion protein partner well. With it we can construct a composite part with better stability and applicability. Two sequences of proteins could be fused with their corresponding fragments at the same time and it can dramatically simplify the cloning process.
+Special designs were taken to optimize the applicability and adaptive of such parts.We disassembled the sequence of IgG and fused it with glucagon to get the recombinant glucagon-IgG-Fc.HA Tag was added to N-terminal of Trim21.The two functional modules of the part (glucagon-IgG-Fc and Trim 21) were connected by the linker GGGGS,one of the best fold breaking linkers to expose fusion protein partner well.
 https://static.igem.org/mediawiki/parts/6/61/BBa_K3064024.png
 <B/r>Figure 1.Representation of the function of the composite part.