Team:BM-AMU/Favorite Part

Team:BM-AMU- 2019.igem.org





Favorite Part

This year,BM-AMU submitted a new composite part(BBa_K3120020), which is constituted by CAG promoter(BBa_K2217013)+Twist1-ERT2(BBa_K3120014)+IRES(BBa_K3120010)+Zeocin ORF(BBa_K076016).

Based on the original plasmid pPYIZ.We established pPYIZ-hTWIST1-Ert2, which was 7728bp.In the plasmid,the CAG promoter activated the expression of Twist1-ERT2. We also added Zeocin ORF for the selection of cell lines which imported Twist1-ERT2 stably.

This is the pattern of our plasmid:

part

In this process,tamoxifen induction system was of great importance.The system regulates gene function at the protein level. In the absence of tamoxifen,Twist1-ERT2 locates in the cytoplasm. When tamoxifen is present,it binds with the ERT2,exposing the nuclear localization signal. Guided by the nuclear localization signal,the Twist1-ERT2-tamoxifen complex translocates to the nucleus,in which Twist1 executes its function.

When we added different concentrations of tamoxifen or treated with tamoxifen for different duration,the progress of EMT would be different.In that situation,the corresponding dose-effect relationship and duration-effect relationship could be obtained.

In cell line WA09-E1 (clone 1),transfected plasmids with lipo2000 liposome and added Zeocin(10 ug/ml) to culture medium.Tamoxifen concentration gradient was 0,2.5,5,7.5,10 (ug/ml) for 3 days. Time gradient were treated with Tamoxifen7.5 micrograms/ml for 1,2 and 3 days,respectively. Then Collected samples and detected the mRNA expression of EMT-related genes by qPCR.

More details in our protocol.

Our experiments proved that tamoxifen induction system works well. Changes in expression of these genes suggest that our tamoxifen induction system can induce TWIST to function successfully,and even cause positive feedback to lead to increased self-expression and at the same time to induce EMT in cells steadily.

Model

Fig 1,mRNA expression of EMT-related genes with different concentrations of tamoxifen (0,2.5ug/ml,5ug/ml,7.5ug/ml,10ug/ml respectively).As the concentration of tamoxifen increased,mRNA expression of Twist1，ZEB1，NCAD，vimentin improved，and the ECAD level went down,which proved that the EMT process was successfully induced.

Model

Fig2,mRNA expression of EMT-related genes with different duration of tamoxifen (0,1d,2d,3d respectively).As the duration of tamoxifen increased,mRNA expression of Twist1，ZEB1，NCAD，vimentin improved，and the ECAD level went down,which proved that the EMT process was successfully induced.