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                <p>[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.
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                 Improvement from iGEM 2024 Team NUDT_CHINA
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                 Contribution from iGEM 2024 Team NUDT_CHINA
 
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                 <p>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.
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                 <p>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.
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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.
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                 <p>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.
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                 <p>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.
 
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                 <p>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.
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                 <p>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.
 
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                <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">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.</p>
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                <p>[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.
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Latest revision as of 10:45, 2 October 2024

TetR-ELK

TetR is a kind of protein that can find and combined with TRE DNA structure domain.ELK is a kind of protein that can activate downstream gene expression after being regulated by phosphorylation

TetR-ELK

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.

1.Pattern diagram

Fig.1 The model diagram TetR-ELK

2.Experiment

2.1 Method

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.

2.2 Result


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

3.Caution

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

Contribution from iGEM 2024 Team NUDT_CHINA

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.

Methods

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.

Results

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.

Reference：

[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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 621
    Illegal NgoMIV site found at 759
    Illegal AgeI site found at 999
  • 1000
    COMPATIBLE WITH RFC[1000]