Part:BBa_K3120020
pCAG+Twist1-ERT2+IRES+Zeocin ORF
Twist1-Ert2 fusion protein was highly expressed by CAG promoter, and when tamoxifen was added, twist1-ert2 fusion protein nucleated and promoted EMT-related genes.The quantitative control of EMT process was realized by controlling tamoxifen concentration.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 629
Illegal NgoMIV site found at 2065
Illegal NgoMIV site found at 2126 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 660
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）.
Our design
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:
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.
The process of experiment
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.
Data and analysis
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.
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