Difference between revisions of "Part:BBa K3704005"
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===Engineering Success=== | ===Engineering Success=== | ||
| + | Build | ||
| + | The coding sequence of GR was cloned by polymerase chain reaction (PCR) and detected by an agarose gel electrician. | ||
| + | [[File:T--ECNUAS-BBa_K3704000 Fig 1a.jpg|500px|thumb|center|]] | ||
| + | [[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. | ||
| + | [[File:T--ECNUAS-BBa_K3704000 Fig 2a.jpg|500px|thumb|center|]] | ||
| + | [[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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Functional verification of the GRE-Fluc reporting system | 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 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). | 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 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). | ||
[[File:T--ECNUAS-BBa_K3704000 Fig 4.png|500px|thumb|center|Figure 1. Relative luciferase activity in the presence of GR agonist (Dexamethasone, Dex) and GR antagonist (Mifepristone, Mife).]] | [[File:T--ECNUAS-BBa_K3704000 Fig 4.png|500px|thumb|center|Figure 1. Relative luciferase activity in the presence of GR agonist (Dexamethasone, Dex) and GR antagonist (Mifepristone, Mife).]] | ||
Revision as of 15:38, 26 October 2020
GRE-F luc
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 which is regulated by GR agonist (increases the fluorescence value) and GR antagonist (reduces the fluorescence value that is stimulated by GR agonist).
Contribution and Biology
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).
Engineering Success
Build The coding sequence of GR was cloned by polymerase chain reaction (PCR) and detected by an agarose 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.
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 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).
