Team:NJU-China/Collaborations
Collaborations
These four parts are artificially designed RNA strands that function as the silencing tool for BCL2 gene’s expression through base-pairing with corresponding mRNAs. BCL2 protein is anti-apoptotic, mainly located in the cytoplasm side of the nuclear membrane, the endoplasmic reticulum and the outer membrane of mitochondria. By muting BCL2 gene’s expression, we can induce more cells’ apoptosis theoretically. We design four sequences and have a DNA synthesis company produce plasmids with codes inserted for each of them.
SYSU-Software Team
As we showed our concern to people’s attitude towards obesity, the SYSU-Software helped us record emoji netizens would use when netizens talk about weight-related topics through web-crawling. Their work provided us with interesting data about netizens’ true reaction to weight-loss and enabled us to cater to ordinaries’ need.
CPU_China
CPU_China were very willing to help us, and they designed two other BCL2 siRNA (si-RNA 3 and 4 ) after we designed two.
We helped the CPU_CHINA team to detect the expression level of Myc-CAR-CD20 and IL-17RA-SynNotch with Western blot. Flag-FOXP3-Jurkat cells expressed the proteins after transfection by electroporation. The result was shown in the figure. We also provided them with human IL-17RA antibodies as one of our collaborations.
our siRNA ( 1&2 is designed by us and 3&4 is designed by them)
NUDT_China
NUDT_China helped us to perform nanoparticle tracking analysis (NTA) to have a more precise determination of the quantity and size of secreted exosomes. They did a supereminent experiment for us and we collected the perfect data we long for.
We constructed a dual-luciferase miRNA target expression plasmid and performed a dual luciferase assay, to help NUDT_CHINA evaluating the microRNA (miRNA) binding ability of miRNA binding sequences.
The pmirGLO Dual-Luciferase miRNA Target Expression Vector was stored in our laboratory plasmid library, and two types of the target sequences (perfectly complementary or not, abbreviated as p and np) of miR-654 were provided by team NUDT_CHINA.
The target sites (each includes eight repeats of the target sequence) were synthesized and inserted into pmirGLO Vector respectively through enzyme digestion and ligation. Then a dual-luciferase miRNA target expression plasmid was constructed.
After that, the purified plasmids were transfected into HEK293T cells to test their functions. Twenty-four hours after transfection with the plasmids, cells were analyzed for luciferase activity using the Dual Luciferase Reporter Gene Assay Kit (Beyotime, RG027) and a Thermo Scientific™ Fluoroskan Ascent™ FL. Normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) for each construct was compared to that of the pmirGLO Vector no-insert control. For each transfection, luciferase activity was averaged from three replicates.
The pmirGLO Dual-Luciferase miRNA Target Expression Vector was stored in our laboratory plasmid library, and two types of the target sequences (perfectly complementary or not, abbreviated as p and np) of miR-654 were provided by team NUDT_CHINA.
The target sites (each includes eight repeats of the target sequence) were synthesized and inserted into pmirGLO Vector respectively through enzyme digestion and ligation. Then a dual-luciferase miRNA target expression plasmid was constructed.
After that, the purified plasmids were transfected into HEK293T cells to test their functions. Twenty-four hours after transfection with the plasmids, cells were analyzed for luciferase activity using the Dual Luciferase Reporter Gene Assay Kit (Beyotime, RG027) and a Thermo Scientific™ Fluoroskan Ascent™ FL. Normalized firefly luciferase activity (firefly luciferase activity/Renilla luciferase activity) for each construct was compared to that of the pmirGLO Vector no-insert control. For each transfection, luciferase activity was averaged from three replicates.
Mechanism and validation of pmirGLO-UTR (p or np) plasmid
(A) Mechanism of action of the pmirGLO-UTR (p or np) plasmid. (B) Results of dual-luciferase reporter assay. The abbreviation “np” stands for cells transfected with pmirGLO-UTR (np), the abbreviation “p” stands for cells transfected with pmirGLO-UTR (p), the abbreviation “control” stands for cells transfected with pmirGLO, the abbreviation “nt” stands for non-transfected cells, the abbreviation “blank” stands for no cells were analyzed,** p < 0.01.
