Difference between revisions of "Part:BBa K3704004"
 
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===Contribution and Biology===
 
===Contribution and Biology===
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Composite part BBa_K3704005 consists of a coding sequence of firefly luciferase under the control of glucocorticoid response element (GRE). Luciferase can catalyze the oxidation of luciferin to oxyluciferin. During the oxidation of luciferin, it will emit bioluminescence. Glucocorticoid receptor (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. BBa_K3704001 forms a reporting system that is regulated by GR agonist (increases the fluorescence value) and GR antagonist (reduces the fluorescence value that is stimulated by GR agonist).
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Composite part BBa_K3704004 consists of a coding sequence of firefly luciferase under the control of glucocorticoid response element (GRE). Luciferase can catalyze the oxidation of luciferin to oxyluciferin. During the oxidation of luciferin, it will emit bioluminescence. Glucocorticoid receptor (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. BBa_K3704001 forms a reporting system that is regulated by GR agonist (increases the fluorescence value) and GR antagonist (reduces the fluorescence value that is stimulated by GR agonist).
  
 
===Engineering Success===
 
===Engineering Success===

Latest revision as of 20:42, 27 October 2020


GR-GRE-F luc-R luc

BBa_K3704004 is an experimental platform to screen small molecules GR antagonists against T2DM for the treatment of T2DM diabetes. Diabetes Mellitus (DM) is a metabolic disease characterized by elevated blood sugar caused by the long-term effects of genetic and environmental factors. According to different pathogenesis, DM mainly includes insulin-dependent type 1 diabetes (Type 1 Diabetes). Mellitus, T1DM), non-insulin-dependent type 2 diabetes (Type 2 Diabetes Mellitus, T2DM), and gestational diabetes (Gestational Diabetes Mellitus, GDM). Among them, T2DM patients account for more than 90% of DM patients. The pathogenesis is mainly the relative lack of insulin caused by peripheral tissue insulin resistance or pancreatic β-cell function defects. Blood sugar cannot be converted into the body's energy requirements, resulting in increased blood sugar. With the improvement of living standards, T2DM is showing an increasing trend and has become a major disease that seriously threatens human health. Glucocorticoid receptor (GR) is one of the members of the metabolism-related nuclear receptor family. It is expressed in many tissues and mainly mediates the physiological effects of glucocorticoids. In diabetic model mice and diabetic patients, the content of glucocorticoid increases, which acts on the glucocorticoid receptor to promote gluconeogenesis and increase blood sugar. In the development of anti-T2DM drugs, GR antagonists have been reported to improve blood glucose levels by reducing gluconeogenesis. Therefore, we established this experimental platform to screen candidate drugs for the treatment of T2DM diabetes.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3025
    Illegal BamHI site found at 1305
    Illegal BamHI site found at 1458
    Illegal BamHI site found at 1572
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1870
    Illegal AgeI site found at 1158
  • 1000
    COMPATIBLE WITH RFC[1000]

Contribution and Biology

Composite part BBa_K3704004 consists of a coding sequence of firefly luciferase under the control of glucocorticoid response element (GRE). Luciferase can catalyze the oxidation of luciferin to oxyluciferin. During the oxidation of luciferin, it will emit bioluminescence. Glucocorticoid receptor (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. BBa_K3704001 forms a reporting system that is regulated by GR agonist (increases the fluorescence value) and GR antagonist (reduces the fluorescence value that is stimulated by GR agonist).

Engineering Success

Functional verification of the GRE-Fluc reporting system In order to test the function of the 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 1. Relative luciferase activity in the presence of GR agonist (Dexamethasone, Dex) and GR antagonist (Mifepristone, Mife).