Team:BM-AMU/Parts

Team:BM-AMU- 2019.igem.org

Overview

This year, we designed 21 parts, and all of them had been submitted to the iGEM Registry following the BioBricks assembly standard as described by RFC10.

Here is a brief description of our parts. If you want to get more detailed information about our parts, we strongly recommend you visit the corresponding parts pages on the iGEM BioBricks Registry.

PARTS TABLE

<groupparts>iGEM19 BM-AMU</groupparts>

Favorite Part

BBa_K3120020

This year，our team submitted 21 new composite parts, we like BBa_K3120020 most. And we also improved quantitative characterization data of it.

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.

In this process, Tamoxifen induction system was of great importance. In the absence of tamoxifen, Twist1-ERT2 located 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. Click herefor more details.

Improve

The Tet system, in which gene expression is stringently controlled by tetracycline (Tc) or its derivative doxycycline (Dox), is the most widely used regulatory circuit. This system is based on the sequence-specific, high-affinity binding of the Escherichia coli Tet repressor protein (TetR) to the tet operator (tetO) DNA sequence. Tc or dox binds to TetR and triggers a conformational change that prevents the repressor protein from binding to tetO. Group USTC and Group Greece have submitted tetR ( BBa_I732080 ) and rtTA ( BBa_K2364002 ) respectively. Unfortunately, The results of the FACS illustrates that without induction with doxycycline, GFP is still expressed. This part, tetR-krab ( BBa_K3120013), is our improvement on the original part. A chimeric transrepressor protein was engineered by fusing the KRAB domain of human Kox1 to the TetR. We hope to get a Tet-On system with low background activity in the absence of dox and high activity in the presence of the effector. To investigate DOX-dependent efficiency of the combined activator strategy, TetO-GFP cell line was constructed in 293-FT cells. Finally, relative fluorescence expression was detected by flow cytometry. Click herefor more details.