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

TetR, also known as tetracycline inhibitory factor, can recognize and bind to the TetO DNA domain. The seven repeated TetO domains are also known as TCR. ELK1 is a protein that can activate downstream gene expression after phosphorylation regulation. Previous studies have cascaded TetR-ELK1 heterozygous transcription factors into the MAPK natural signaling pathway to construct insulin sensors1. When the insulin receptor (INRS) is stimulated by insulin, INRS phosphorylates insulin receptor substrate (IRS) and ultimately activates MAPK. MAPK phosphorylates ELK1 and initiates downstream gene expression. In the BBa_K3734019 file of iGEM21_CSU_CINA in 2021, they activated the expression of downstream fluorescent reporter gene mCherry using TetR-ELK1 and observed the expression of fluorescence under laser confocal microscopy. On this basis, we used SEAP to test the sensitivity and reporting intensity of TetR-ELK1 to insulin.

All the plasmid of P_EF1a-INSR, TetR-ELK1 and TCE-SEAP were co-transfected into HEK-293T cells with the ratio of 1: 1: 1 (both transfected with 100ng), after which cells were stimulated by 0nM, 1nM, 10nM and 20nM four different concentrations of insulin respectively. Data was collected 24h after stimulation.

After 0, 10, and 20nm insulin stimulation, the SEAP activity of transfected cells was 26-27 times higher than that of unstimulated cells. This proved that The TetR-ELK1 pathway can strongly respond to low insulin stimulation. Figures below indicated quantitative data of the experiments.

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 1nm, 10nm, and 20nm after 24 hours to detect the SEAP activity; 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.