Difference between revisions of "Part:BBa K3734019"

TetR can recognize and combine with TRE, it is a important part of Tet-off system. Without tetracycline, TetR can combine with TRE. With the presence of tetracycline, the combination of TetR and TRE will be blocked and the Tet-off system will be shut down. ELK is generally dormant, when INSR receive insulin and activate MAPK, ELK will be phophated and activated, then ELK is able activate expression of target gene downstream TRE.

Fig.1 The model diagram TetR-ELK

After TetR binds to TRE, phosphorylation ELK activates downstream reporting gene mCherry expression, and we observe the expression with laser confocal microscope.

We also use ERK antibodies to detect phosphorylation of ERK1/ERK2 in the phosphorylation pathway through Western Blot.

Fig.2 Under Laser confocal microscopy, fluorescence of mCherry expression downstream of Tet-Off system

Fig.3 ERK phosphorylation changes with different insulin treatment

Fig.4 ERK phosphorylation changes with different insulin treatment

Despite the length and the complication of phosphorylation pathway, the phosphorylation pathway of protein is a very short process, it is usually completed within minutes even seconds. The time of cracking the cells and collecting protein must be controlled precisely when detecting

The tetR-ELK1 was originally desiged as a signal transducer rewiring the activation of MAPK signaling pathway to into the transcriptional activation of tetO7 promoters [1]. The team iGEM21_CSU_CINA in 2021 have already shown the change of MAPK-ERK pathway with insulin stimulation, we hereby further chacterize the transcriptional activation of TetR-ELK1 in response of insulin stimulation.

HEK-293T cells were co-transfected with three plasmids (in 1:1:1 ratio) carrying either insulin receptor (INSR), TetR-ELK1 and TCE-SEAP cassette respectively. Cells were stimulated with either 0nM, 1nM, 10nM or 20nM insulin at 6 hours post transfection. SEAP level in the cell culture medium was measured 24h after stimulation.

As shown in Figure 5, insulin stimulation resulted in an approximately 26-27-fold activation of SEAP production compared to the unstimulated cells. This result provided a quantitative characterization on how TetR-ELK1 could respond to insulin stimulation.

Fig.5 Functional validation of TetR-ELK1 pathway under insulin stimulation. HEK293T cells were transfected with P_EF1a-INRS, TetR-ELK1 and TCE-SEAP in a 1:1:1 ratio and stimulated with insulin at concentrations of 1 nM, 10 nM, and 20 nM 6 hours post transfection. SEAP level was measured at 24 hours after insulin stimulation; data shows mean±SD, n=3 independent experiments.

[1]Haifeng Ye, Mingqi Xie, Shuai Xue.Self-adjusting synthetic gene circuit for correcting insulin resistance[J].Nat Biomed Eng.2017 Jan;1(1):0005.