Team:BM-AMU/Improve

Team:BM-AMU- 2019.igem.org

Improve

Abstract

This year, we got an improved part (BBa_K3120013)based on the original part (BBa_K2364002) .

Background

Regulated Mammalian Expression Systems enable the expression of interest gene(s) to be strictly controlled in a quantitative and temporal manner. They are widely used in human life science research, including functional genomics, gene therapy, animal diseases models and biopharmaceutical industry. Tet-On system is based on the sequence-specific, high-affinity binding of the Tet repressor protein (TetR) to the tet operator (tetO) DNA sequence. Expression from a target transgene is dependent on the activity of an inducible transcriptional activator, whose expression can be regulated both reversibly and quantitatively by exposing to varying concentrations of tetracycline (Tc), or Tc derivatives such as doxycycline (Dox). Transcription is active in the presence of Dox, since dox binds to TetR and triggers a conformational change that prevents the repressor protein from binding to tetO.

Previous Parts

The Tet-On system is based on a reverse tetracycline-controlled transactivator, rtTA. rtTA is a fusion protein comprised of the TetR repressor and the VP16 transactivation domain; however, a four amino acid change in the tetR DNA binding moiety alters rtTA's binding characteristics such that it can only recognize the tetO sequences in the tetracycline-responsive promoter element (TRE) of the target transgene in the presence of the Dox effector. Thus, in the Tet-On system, transcription of the TRE-regulated target gene is stimulated by rtTA only in the presence of Dox. TetR ( BBa_I732080 ) and rtTA ( BBa_K2364002 ) have been submitted by Group USTC and Group Greece respectively.

Improved Parts

We designed a new parttetR-krab ( BBa_K3120013 )as the improvement of tetR. KRAB is a 75 aa domain found in about one third of the several hundreds of human zinc finger proteins and is located in the amino terminal end of proteins that contain Krüppel-class zinc fingers in their carboxy termini. A chimeric transrepressor protein was engineered by fusing the KRAB domain of human Kox1 to the TetR.

Results

To compare DOX-dependent efficiency of tetR, rtTA and tet-Krab, these parts were constructed into 293FT cell lines seperately. Then, equal amount of 293T were seeded in a 6-well plate and transfected with TetO-GFP vector. GFP intensity was detected at 5, 7, 9 days post transfection by flow cytometry. On the day 9, DOX (1 μg/μL) was added to initiate downstream gene expression, and relative GFP expression was measured 5 days later . Experimental details can be found in our protocol.

Gene expression in Tet-On system requires with low background activity in the absence of dox and high activity in the presence of the effector. The results showed GFP expression induced by Dox were similar in three tet-on systems. However, tetR also gave a strong expression of GFP even without doxycycline induction, with rtTA was slightly weaker. The improved part tetR-krab can significantly prevent the leakage of GFP expression.