Team:SZU China/Result
Result
Renilla Luciferase assay in three plasmids system
Three recombinant plasmids were generated and transiently transfected into a human bladder cancer cell line T24.
To test the working efficiency of our orthogonal system, we devided the cells into two groups. One has Ack in the culture medium and another does not.
Luciferase assays were performed using luminometer. Since Ack is essential for our system, we speculated that Renilla Luciferase(Rluc), the output gene in the circuits can be selectively expressed in Experiment group with Ack. As shown in Fig. 1, the activity of Renilla Luciferase(RLUC) varied widely between the two groups, and the circuit only demonstrated significant activity in the experimental group as expected. Therefore, our orthogonal system is verified to be in good condition and can work efficiently.
Luciferase assay in two plasmids system
hTERT and hUPII promoters are cancer specific promoter and bladder specific promoter, respectively. By combining the two promoters into the design, we supposed that the constructed circuits have the ability to selectively identify bladder cancer cells, in which telomerase and uroplakins are both expressed.
To test this hypothesis, we used the luciferase reporter gene as the output of the circuit and tested the luciferase activity in HFC, which is short for human fiber epithelial cell, Hela, a cervical carcinoma cell line and 5637, a bladder cancer cell line. Also, we reconstructed our system in two plasmids to test if two plasmids system can perform higher efficiency.
The result (Fig.2) shows that the activity of LUC in 5637 with Ack was about two times as high as that in 5637 without Ack and three times as high as that in Hela, while it could not be measured in HFC. We can learn from this result that this system is perfectly safe for normal cells, with no luciferase being detected in HFC. However, compared with three plasmids system, whose RLUC activity of experimental group is about 7 times as high as that of control group, the working efficiency of our orthogonal system in two plasmids is much lower. So we had better construct our system in two plasmids to express higher level of therapeutic gene in bladder cancer cells and reduce its expression in other cell types to increase its specificity.
Green fluorescent light measurement in two and three plasmids system
To further verifying the working efficiency of the unnatural amino acid orthogonal system, we replaced the amber mutated output gene in former plasmids to amber mutated GFP. The plasmid psiCHECKTM2-CMV-GFP was used as a positive control to monitor transfection and expression efficiency. The two plasmids and three plasmids system being constructed by GFP recombinant plasmids were transiently transfected into 5637 and T24, both of which are bladder cancer cell line.
Pictures of green fluorescent light produced by target cells were taken by fluorescent microscope. From these pictures (Fig. 3), we can see there is no expression of GFP being detected in no Ack groups while the light intensities are rather high in with Ack groups no matter in T24 or 5637 cell lines. These results indicate that the output gene is expressed only in cells which have Ack in the culture medium.
Conclusion
These findings suggest that the constructed circuit based on AND GATE and UAA orthogonal system can be used to specifically identify bladder cancer cells and express therapeutic gene only when Ack exist. These results may yield a new therapeutic approach for bladder cancer.