Difference between revisions of "Part:BBa K3704000"

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===Engineering Success===
 
===Engineering Success===
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Verify the function GR using a luciferase reporting system
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Build
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In order to test the function of the GR, a recombinant GR producing construct (BBa_K3704002) and a luciferase reporting system (two constructs contain parts BBa_K3704001 and BBa_K3522012, respectively) were co-transfected into HEK293T cells. With the addition of GR agonist (Dexamethasone, Dex) into the cell culture, a significant value of luciferase activity was detected (Figure 1). While, in the presence of Dex and a GR antagonist (Mifepristone, Mife), the luciferase activity reduced to a value similar to the blank control (DMSO).  
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[[File:T--ECNUAS-BBa_K3704000 Fig 4.png|400px|thumb|center|Figure 1. Relative luciferase activity in the presence of GR agonist (Dexamethasone, Dex) and GR antagonist (Mifepristone, Mife).]]
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The coding sequence of GR was cloned by polymerase chain reaction (PCR) and detected by an agarose gel electrician.
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[[File:T--ECNUAS-BBa_K3704000 Fig 1a.jpg|500px|thumb|center|]]
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[[File:T--ECNUAS-BBa_K3704000 Fig 1b.jpg|500px|thumb|center|Fig 1. The detection of the GR fragments by a garose gel electrician.]]
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The DNA molecular weight of the GR fragments was correct. We cut out positive bands for gel extraction to obtain the GR fragment.
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Then, we ligated the GR fragment and plasmid vector PCL- and transformed them into E.coli DH5α competent cells. After culturing transformers overnight, we picked some single clones to enlarge culture and extracted the plasmid. Finally, we testified the plasmid by sanger sequencing. 
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[[File:T--ECNUAS-BBa_K3704000 Fig 2a.jpg|500px|thumb|center|]]
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[[File:T--ECNUAS-BBa_K3704000 Fig 2b.jpg|500px|thumb|center|Fig2. The sequence alignment of the pCL-GR plasmid.Pairwise sequence alignment showed we conducted the plasmid pCL-GR success.]]
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Test
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In order to test the function of the GR and GRE-Fluc reporting system, GRE-Fluc containing plasmid and another two plasmids which produce GR (part BBa_K3704002) and Renilla luciferase (BBa_K3522012, used as an internal reference gene to provide a unified baseline for experiments), respectively, were co-transfected into HEK293T cells. With addition of GR agonist (Dexamethasone, Dex) into the cell culture, significant value of luciferase activity was detected (Figure 1). While, in the presence of Dex and a GR antagonist (Mifepristone, Mife), the luciferase activity reduced to a value similar to the blank control (DMSO).  
 +
 
 +
[[File:T--ECNUAS-BBa_K3704000 Fig 4.png|500px|thumb|center|Figure 3. Relative luciferase activity in the presence of GR agonist (Dexamethasone, Dex) and GR antagonist (Mifepristone, Mife).]]
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Conclusion
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The experiment result shows that we have built a dual fluorescent reporter gene screening platform for GR anti-type 2 diabetes lead compounds. It provides a material basis for high-throughput screening of compounds that target antagonistic GR and anti-T2DM.

Revision as of 15:43, 26 October 2020


GR

BBa_K3704000 is a protein domain of the glucocorticoid receptor (PubMed:27120390). Has a dual mode of action: a transcription factor that binds to glucocorticoid response elements (GRE), both for nuclear and mitochondrial DNA, and as a modulator of other transcription factors. Affects inflammatory responses, cellular proliferation, and differentiation in target tissues. Involved in chromatin remodeling (PubMed:9590696). Plays a role in rapid mRNA degradation by binding to the 5' UTR of target mRNAs and interacting with PNRC2 in a ligand-dependent manner that recruits the RNA helicase UPF1 and the mRNA-decapping enzyme DCP1A, leading to RNA decay (PubMed:25775514). Could act as a coactivator for STAT5-dependent transcription upon growth hormone (GH) stimulation and could reveal an essential role of hepatic GR in the control of body growth (By similarity).

Biology and Contribution

Composite part BBa_K3704000 is the coding sequence of the glucocorticoid receptor (GR). GR belongs to the steroid hormone receptor subclass of nuclear receptors, which controls physiological processes by activating and inhibiting specific target genes (PMID: 15379669). The ligand-activated receptor dimer activates gene expression by binding to GRE in the promoter region of the glucocorticoid regulatory gene.

Engineering Success

Build

The coding sequence of GR was cloned by polymerase chain reaction (PCR) and detected by an agarose gel electrician.

T--ECNUAS-BBa K3704000 Fig 1a.jpg
Fig 1. The detection of the GR fragments by a garose gel electrician.

The DNA molecular weight of the GR fragments was correct. We cut out positive bands for gel extraction to obtain the GR fragment.

Then, we ligated the GR fragment and plasmid vector PCL- and transformed them into E.coli DH5α competent cells. After culturing transformers overnight, we picked some single clones to enlarge culture and extracted the plasmid. Finally, we testified the plasmid by sanger sequencing.

T--ECNUAS-BBa K3704000 Fig 2a.jpg
Fig2. The sequence alignment of the pCL-GR plasmid.Pairwise sequence alignment showed we conducted the plasmid pCL-GR success.

Test

In order to test the function of the GR and GRE-Fluc reporting system, GRE-Fluc containing plasmid and another two plasmids which produce GR (part BBa_K3704002) and Renilla luciferase (BBa_K3522012, used as an internal reference gene to provide a unified baseline for experiments), respectively, were co-transfected into HEK293T cells. With addition of GR agonist (Dexamethasone, Dex) into the cell culture, significant value of luciferase activity was detected (Figure 1). While, in the presence of Dex and a GR antagonist (Mifepristone, Mife), the luciferase activity reduced to a value similar to the blank control (DMSO).

Figure 3. Relative luciferase activity in the presence of GR agonist (Dexamethasone, Dex) and GR antagonist (Mifepristone, Mife).

Conclusion

The experiment result shows that we have built a dual fluorescent reporter gene screening platform for GR anti-type 2 diabetes lead compounds. It provides a material basis for high-throughput screening of compounds that target antagonistic GR and anti-T2DM.